Production and Characterization of a Novel Exopolysaccharide from Ramlibacter tataouinensis.

The current study examines the desiccation-resistant Ramlibacter tataouinensis TTB310T as a model organism for the production of novel exopolysaccharides and their structural features. This bacterium is able to produce dividing forms of cysts which synthesize cell-bound exopolysaccharide. Initial experiments were conducted on the enrichment of cyst biomass for exopolysaccharide production under batch-fed conditions in a pilot-scale bioreactor, with lactate as the source of carbon and energy. The optimized medium produced significant quantities of exopolysaccharide in a single growth phase, since the production of exopolysaccharide took place during the division of the cysts. The exopolysaccharide layer was extracted from the cysts using a modified trichloroacetic acid method. The biochemical characterization of purified exopolysaccharide was performed by gas chromatography, ultrahigh-resolution mass spectrometry, nuclear magnetic resonance, and Fourier-transform infrared spectrometry. The repeating unit of exopolysaccharide was a decasaccharide consisting of ribose, glucose, rhamnose, galactose, mannose, and glucuronic acid with the ratio 3:2:2:1:1:1, and additional substituents such as acetyl, succinyl, and methyl moieties were also observed as a part of the exopolysaccharide structure. This study contributes to a fundamental understanding of the novel structural features of exopolysaccharide from a dividing form of cysts, and, further, results can be used to study its rheological properties for various industrial applications.

Unraveling the interplay between root exudates, microbiota, and rhizosheath formation in pearl millet.

BACKGROUND: The rhizosheath, a cohesive soil layer firmly adhering to plant roots, plays a vital role in facilitating water and mineral uptake. In pearl millet, rhizosheath formation is genetically controlled and influenced by root exudates. Here, we investigated the impact of root exudates on the microbiota composition, interactions, and assembly processes, and rhizosheath structure in pearl millet using four distinct lines with contrasting soil aggregation abilities. RESULTS: Utilizing 16S rRNA gene and ITS metabarcoding for microbiota profiling, coupled with FTICR-MS metabonomic analysis of metabolite composition in distinct plant compartments and root exudates, we revealed substantial disparities in microbial diversity and interaction networks. The ß-NTI analysis highlighted bacterial rhizosphere turnover driven primarily by deterministic processes, showcasing prevalent homogeneous selection in root tissue (RT) and root-adhering soil (RAS). Conversely, fungal communities were more influenced by stochastic processes. In bulk soil assembly, a combination of deterministic and stochastic mechanisms shapes composition, with deterministic factors exerting a more pronounced role. Metabolic profiles across shoots, RT, and RAS in different pearl millet lines mirrored their soil aggregation levels, emphasizing the impact of inherent plant traits on microbiota composition and unique metabolic profiles in RT and exudates. Notably, exclusive presence of antimicrobial compounds, including DIMBOA and H-DIMBOA, emerged in root exudates and RT of low aggregation lines. CONCLUSIONS: This research underscores the pivotal influence of root exudates in shaping the root-associated microbiota composition across pearl millet lines, entwined with their soil aggregation capacities. These findings underscore the interconnectedness of root exudates and microbiota, which jointly shape rhizosheath structure, deepening insights into soil-plant-microbe interactions and ecological processes shaping rhizosphere microbial communities. Deciphering plant-microbe interactions and their contribution to soil aggregation and microbiota dynamics holds promise for the advancement of sustainable agricultural strategies. Video Abstract.

Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation.

BACKGROUND: Deinococcus deserti VCD115 has been isolated from Sahara surface sand. This radiotolerant bacterium represents an experimental model of choice to understand adaptation to harsh conditions encountered in hot arid deserts. We analysed the soluble proteome dynamics in this environmentally relevant model after exposure to 3 kGy gamma radiation, a non-lethal dose that generates massive DNA damages. For this, cells were harvested at different time lapses after irradiation and their soluble proteome contents have been analysed by 2-DE and mass spectrometry. RESULTS: In the first stage of the time course we observed accumulation of DNA damage response protein DdrB (that shows the highest fold change ~11), SSB, and two different RecA proteins (RecAP and RecAC). Induction of DNA repair protein PprA, DNA damage response protein DdrD and the two gyrase subunits (GyrA and GyrB) was also detected. A response regulator of the SarP family, a type II site-specific deoxyribonuclease and a putative N-acetyltransferase are three new proteins found to be induced. In a more delayed stage, we observed accumulation of several proteins related to central metabolism and protein turn-over, as well as helicase UvrD and novel forms of both gyrase subunits differing in terms of isoelectric point and molecular weight. CONCLUSIONS: Post-translational modifications of GyrA (N-terminal methionine removal and acetylation) have been evidenced and their significance discussed. We found that the Deide_02842 restriction enzyme, which is specifically found in D. deserti, is a new potential member of the radiation/desiccation response regulon, highlighting the specificities of D. deserti compared to the D. radiodurans model.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

Phytobeneficial traits of rhizobacteria under the control of multiple molecular dialogues.

Pseudomonads play crucial roles in plant growth promotion and control of plant diseases. However, under natural conditions, other microorganisms competing for the same nutrient resources in the rhizosphere may exert negative control over their phytobeneficial characteristics. We assessed the expression of phytobeneficial genes involved in biocontrol, biostimulation and iron regulation such as, phlD, hcnA, acdS, and iron-small regulatory RNAs prrF1 and prrF2 in Pseudomonas brassicacearum co-cultivated with three phytopathogenic fungi, and two rhizobacteria in the presence or absence of Brassica napus, and in relation to iron availability. We found that the antifungal activity of P. brassicacearum depends mostly on the production of DAPG and not on HCN whose production is suppressed by fungi. We have also shown that the two-competing bacterial strains modulate the plant growth promotion activity of P. brassicacearum by modifying the expression of phlD, hcnA and acdS according to iron availability. Overall, it allows us to better understand the complexity of the multiple molecular dialogues that take place underground between microorganisms and between plants and its rhizosphere microbiota and to show that synergy in favour of phytobeneficial gene expression may exist between different bacterial species.

Complete genome sequence of a beneficial plant root-associated bacterium, Pseudomonas brassicacearum.

To shed light on the genetic equipment of the beneficial plant-associated bacterium Pseudomonas brassicacearum, we sequenced the whole genome of the strain NFM421. Its genome consists of one chromosome equipped with a repertoire of factors beneficial for plant growth. In addition, a complete type III secretion system and two complete type VI secretion systems were identified. We report here the first genome sequence of this species.

Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments.

In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.

Evolutionary history expands the range of signaling interactions in hybrid multikinase networks.

Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.

Mutagenic lesion bypass and two functionally different RecA proteins in Deinococcus deserti.

RecA is essential for extreme radiation tolerance in Deinococcus radiodurans. Interestingly, Sahara bacterium Deinococcus deserti has three recA genes (recA(C), recA(P1), recA(P3)) that code for two different RecA proteins (RecA(C), RecA(P)). Moreover, and in contrast to other sequenced Deinococcus species, D. deserti possesses homologues of translesion synthesis (TLS) DNA polymerases, including ImuY and DnaE2. Together with a lexA homologue, imuY and dnaE2 form a gene cluster similar to a widespread RecA/LexA-controlled mutagenesis cassette. After having developed genetic tools, we have constructed mutant strains to characterize these recA and TLS polymerase genes in D. deserti. Both RecA(C) and RecA(P) are functional and allow D. deserti to survive, and thus repair massive DNA damage, after exposure to high doses of radiation. D. deserti is mutable by UV, which requires ImuY, DnaE2 and RecA(C), but not RecA(P). RecA(C), but not RecA(P), facilitates induced expression of imuY and dnaE2 following UV exposure. We propose that the extra recA(P1) and recA(P3) genes may provide higher levels of RecA protein for efficient error-free repair of DNA damage, without further increasing error-prone lesion bypass by ImuY and DnaE2, whereas limited TLS may contribute to adaptation to harsh conditions by generating genetic variability.

Light on the cell cycle of the non-photosynthetic bacterium Ramlibacter tataouinensis.

Ramlibacter tataouinensis TTB310, a non-photosynthetic betaproteobacterium isolated from a semi-arid region of southern Tunisia, forms both rods and cysts. Cysts are resistant to desiccation and divide when water and nutrients are available. Rods are motile and capable of dissemination. Due to the strong correlation between sunlight and desiccation, light is probably an important external signal for anticipating desiccating conditions. Six genes encoding potential light sensors were identified in strain TTB310. Two genes encode for bacteriophytochromes, while the four remaining genes encode for putative blue light receptors. We determined the spectral and photochemical properties of the two recombinant bacteriophytochromes RtBphP1 and RtBphP2. In both cases, they act as sensitive red light detectors. Cyst divisions and a complete cyst-rod-cyst cycle are the main processes in darkness, whereas rod divisions predominate in red or far-red light. Mutant phenotypes caused by the inactivation of genes encoding bacteriophytochromes or heme oxygenase clearly show that both bacteriophytochromes are involved in regulating the rod-rod division. This process could favor rapid rod divisions at sunrise, after dew formation but before the progressive onset of desiccation. Our study provides the first evidence of a light-based strategy evolved in a non-photosynthetic bacterium to exploit scarse water in a desert environment.

The cyst-dividing bacterium Ramlibacter tataouinensis TTB310 genome reveals a well-stocked toolbox for adaptation to a desert environment.

Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical "cyst-like" cells ("cyst-cyst" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.

ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis.

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.