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The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium / Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium / Methylorubrum strains for advantageous agricultural applications. A point mutation in the phosphoribosylpyrophosphate synthetase gene is found to rewire C1 metabolism and thereby enhance colonization capabilities and growth-promoting effects in plants. The effect of this mutation is prevalent across Methylobacterium strains.

Background Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). Results This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography—tandem mass spectrometry (HPLC–MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL −1 for total CKs, and 0.46 to 82.16 pmol mL −1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium . Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. Conclusions To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M . extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

An aerobic, Gram-stain-negative, motile rod bacterium, designated as SYSU BS000021 T , was isolated from a black soil sample in Harbin, Heilongjiang province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Methylobacterium , and showed the highest sequence similarity to Methylobacterium segetis KCTC 62267  T (98.51%) and Methylobacterium oxalidis DSM 24028  T (97.79%). Growth occurred at 20–37℃ (optimum, 28 °C), pH 6.0–8.0 (optimum, pH 7.0) and in the presence of 0% (w/v) NaCl. Polar lipids comprised of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminolipid and one unidentified polar lipid. The major cellular fatty acids (> 5%) were C 18:0 and C 18:1 ω 7 c and/or C 18:1 ω 6 c . The predominant respiratory quinone was Q-10. The genomic G + C content was 68.36% based on the whole genome analysis. The average nucleotide identity (≤ 83.5%) and digital DNA–DNA hybridization (≤ 27.3%) values between strain SYSU BS000021 T and other members of the genus Methylobacterium were all lower than the threshold values recommended for distinguishing novel prokaryotic species. Based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, strain SYSU BS000021 T represents a novel species of the genus Methylobacterium , for which the name Methylobacterium nigriterrae sp. nov. is proposed. The type strain of the proposed novel species is SYSU BS000021 T (= GDMCC 1.3814  T  = KCTC 8051  T ).

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES ('MP') medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.

Epistasis has substantial impacts on evolution, in particular, the rate of adaptation. We generated combinations of beneficial mutations that arose in a lineage during rapid adaptation of a bacterium whose growth depended on a newly introduced metabolic pathway. The proportional selective benefit for three of the four loci consistently decreased when they were introduced onto more fit backgrounds. These three alleles all reduced morphological defects caused by expression of the foreign pathway. A simple theoretical model segregating the apparent contribution of individual alleles to benefits and costs effectively predicted the interactions between them. These results provide the first evidence that patterns of epistasis may differ for within- and between-gene interactions during adaptation and that diminishing returns epistasis contributes to the consistent observation of decelerating fitness gains during adaptation.

The plant phyllosphere constitutes a habitat for numerous microorganisms; among them are members of the genus Methylobacterium . Owing to the ubiquitous occurrence of methylobacteria on plant leaves, they represent a suitable target for studying plant colonization patterns. The influence of the factor site, host plant species, time and the presence of other phyllosphere bacteria on Methylobacterium community composition and population size were evaluated in this study. Leaf samples were collected from Arabidopsis thaliana or Medicago truncatula plants and from the surrounding plant species at several sites. The abundance of cultivable Methylobacterium clearly correlated with the abundance of other phyllosphere bacteria, suggesting that methylobacteria constitute a considerable and rather stable fraction of the phyllosphere microbiota under varying environmental conditions. Automated ribosomal intergenic spacer analysis (ARISA) was applied to characterize the Methylobacterium community composition and showed the presence of similar communities on A. thaliana plants at most sites in 2 consecutive years of sampling. A substantial part of the observed variation in the community composition was explained by site and plant species, especially in the case of the plants collected at the Arabidopsis sites (50%). The dominating ARISA peaks that were detected on A. thaliana plants were found on other plant species grown at the same site, whereas some different peaks were detected on A. thaliana plants from other sites. This indicates that site-specific factors had a stronger impact on the Methylobacterium community composition than did plant-specific factors and that the Methylobacterium –plant association is not highly host plant species specific.

The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negativeMethylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.

This study is aimed to assess the biodegradation of sulfadiazine (SDZ) and characterization of heavy metal resistance in three pure bacterial cultures and also their chemotactic response towards 2-aminopyrimidine. The bacterial cultures were isolated from pig manure, activated sludge and sediment samples, by enrichment technique on SDZ (6 mg L-1). Based on the 16S rRNA gene sequence analysis, the microorganisms were identified within the genera of Paracoccus, Methylobacterium and Kribbella, which were further designated as SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47. The three identified pure bacterial strains degraded up to 50.0, 55.2 and 60.0% of SDZ (5 mg L-1), respectively within 290 h. On the basis of quadrupole time-of-flight mass spectrometry and high performance liquid chromatography, 2-aminopyrimidine and 4-hydroxy-2-aminopyrimidine were identified as the main intermediates of SDZ biodegradation. These bacteria were also able to degrade the metabolite, 2-aminopyrimidine, of the SDZ. Furthermore, SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47 also showed resistance to various heavy metals like copper, cadmium, chromium, cobalt, lead, nickel and zinc. Additionally, all three bacteria exhibited positive chemotaxis towards 2-aminopyrimidine based on the drop plate method and capillary assay. The results of this study advanced our understanding about the microbial degradation of SDZ, which would be useful towards the future SDZ removal in the environment.

Metabolic homeostasis is a central organizing principle of physiology whereby dynamic processes work to maintain a balanced internal state. Highly reactive essential metabolites are ideally maintained at equilibrium to prevent cellular damage. In the facultative methylotrophic bacterium Methylobacterium extorquens PA1, the utilization of one-carbon growth substrates, including methanol, generates formaldehyde as an obligate intermediate. Formaldehyde is highly chemically reactive and capable of damaging various biomolecules, making formaldehyde homeostasis critical during methylotrophic growth. However, homeostatic mechanisms that govern formaldehyde balance, which is readily perturbed upon transitioning to methylotrophic growth substrates, have remained elusive. Here we describe how a formaldehyde-sensing protein EfgA, a formaldehyde-responsive MarR-like regulator TtmR, and lanthanide-mediated methylotrophy together impact formaldehyde balance and one-carbon metabolism more broadly when cells are transitioning to growth on formaldehyde-generating one-carbon sources. We found that cells lacking efgA or ttmR are unable to maintain formaldehyde balance during various carbon source transitions resulting in elevated extracellular formaldehyde concentrations and an extended lag phase. In strains lacking efgA , we showed that inflated intracellular formaldehyde pools were accompanied by decreased cell viability, while the loss of ttmR resulted in the loss of one-carbon metabolites to the extracellular space. Additionally, we found less severe formaldehyde imbalances in the presence of lanthanides, even in the absence of efgA and ttmR. This was partly due to the activation of exaF , a lanthanide-dependent alcohol dehydrogenase that served as an alternative formaldehyde-detoxifying system that lessened the necessity of ttmR for maintaining formaldehyde homeostasis. Overall, our data demonstrated that efgA has a primary role in formaldehyde homeostasis in modulating intracellular formaldehyde pools, while ttmR is secondary, preventing carbon loss to the extracellular space. These results led us to develop a model of formaldehyde homeostasis involving formaldehyde sensing, growth arrest, compartmentalization, and auxiliary detoxification systems. This work deepens our understanding of how physiological factors impact biological formaldehyde homeostasis during transient metabolic imbalances of this universal cellular toxin.