Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

Tol-Pal System and Rgs Proteins Interact to Promote Unipolar Growth and Cell Division in Sinorhizobium meliloti.

Sinorhizobium meliloti is an alphaproteobacterium belonging to the Rhizobiales Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal, and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ, and Pal localized to the growing cell pole in the cell elongation phase and to the septum in predivisional cells, and three Rgs proteins localized to the growing cell pole only. The putative FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of the Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other alphaproteobacterial orders, suggesting a conserved role of these proteins in polar growth.IMPORTANCE Bacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod-shaped bacteria is cell elongation by incorporating new peptidoglycans in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the alphaproteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

Sinorhizobium meliloti, a Slow-Growing Bacterium, Exhibits Growth Rate Dependence of Cell Size under Nutrient Limitation.

Bacterial cells need to coordinate the cell cycle with biomass growth to maintain cell size homeostasis. For fast-growing bacterial species like Escherichia coli and Bacillus subtilis, it is well-known that cell size exhibits a strong dependence on the growth rate under different nutrient conditions (known as the nutrient growth law). However, cell size changes little with slow growth (doubling time of >90 min) for E. coli, posing the interesting question of whether slow-growing bacteria species also observe the nutrient growth law. Here, we quantitatively characterize the cell size and cell cycle parameter of a slow-growing bacterium, Sinorhizobium meliloti, at different nutrient conditions. We find that S. meliloti exhibits a threefold change in its cell size when its doubling time varies from 2 h to 6 h. Moreover, the progression rate of its cell cycle is much longer than that of E. coli, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate. Our study shows that the nutrient growth law holds robustly regardless of the growth capacity of the bacterial species, generalizing its applicability among the bacterial kingdom.IMPORTANCE The dependence of cell size on growth rate is a fundamental principle in the field of bacterial cell size regulation. Previous studies of cell size regulation mainly focus on fast-growing bacterial species such as Escherichia coli and Bacillussubtilis We find here that Sinorhizobium meliloti, a slow-growing bacterium, exhibits a remarkable growth rate-dependent cell size pattern under nutrient limitation, generalizing the applicability of the empirical nutrient growth law of cell size. Moreover, S. meliloti exhibits a much slower speed of cell cycle progression than E. coli does, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate.

Monitoring succinoglycan production in single Sinorhizobium meliloti cells by Calcofluor white M2R staining and time-lapse microscopy.

Here, we describe a simple, non-time consuming and inexpensive method for monitoring of Calcofluor white M2R-binding exopolysaccharides in individual bacterial cells. This method was demonstrated by time-lapse microscopy of succinoglycan-producing cells of the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti. The method is most likely applicable to other bacteria producing ß-(1→3) and ß-(1→4) linked polysaccharides.

Sinorhizobium meliloti Chemotaxis to Multiple Amino Acids Is Mediated by the Chemoreceptor McpU.

The legume symbiont Sinorhizobium meliloti is chemoattracted to compounds exuded by germinating seeds of its host alfalfa. This response is mainly mediated by the S. meliloti chemoreceptor McpU. McpU also has a prominent contribution in sensing a synthetic amino acid (aa) mixture mimicking the amounts and composition observed in seed exudate. Here, we used the hydrogel capillary assay to quantify chemotactic responses of S. meliloti to individual aa exuded by germinating alfalfa seeds and to define the role of McpU in this behavior. S. meliloti exhibited positive chemotaxis responses to all proteinogenic aa, except for aspartate, and to citrulline, cystine, gamma-aminobutyric acid, and ornithine. Wild-type responses were diverse in intensity, while a strain lacking mcpU displayed strongly diminished responses. Differential scanning fluorimetry demonstrated interaction of the purified periplasmic region of McpU (McpU-PR) with the aa, except glutamate and aspartate. We additionally tested organic acids and sugars, but there were no significant interactions with the McpU ligand-binding domain, except for citrate. Using ligand displacement, we confirmed the interaction of McpU-PR with aa representing strong and weak attractants. Our results show that S. meliloti McpU is a broad-range aa receptor mediating differential responses to individual attractants, which does not bind negatively charged aa.

Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle.

A considerable share of bacterial species maintains multipartite genomes. Precise coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. The α-proteobacterium Sinorhizobium meliloti possesses a tripartite genome composed of one chromosome and the megaplasmids pSymA and pSymB. Here, we investigated the spatiotemporal pattern of segregation of these S. meliloti replicons at single cell level. Duplication of chromosomal and megaplasmid origins of replication occurred spatially and temporally separated, and only once per cell cycle. Tracking of FROS (fluorescent repressor operator system)-labelled origins revealed a strict temporal order of segregation events commencing with the chromosome followed by pSymA and then by pSymB. The repA2B2C2 region derived from pSymA was sufficient to confer the spatiotemporal behaviour of this megaplasmid to a small plasmid. Altering activity of the ubiquitous prokaryotic replication initiator DnaA, either positively or negatively, resulted in an increase in replication initiation events or G1 arrest of the chromosome only. This suggests that interference with DnaA activity does not affect replication initiation control of the megaplasmids.

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti.

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.

Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy.

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

Monitoring trehalose uptake and conversion by single bacteria using laser tweezers Raman spectroscopy.

Having the ability to monitor metabolic activity at the scale of single bacterial cells noninvasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell-cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.

The Sinorhizobium meliloti sensor histidine kinase CbrA contributes to free-living cell cycle regulation.

Sinorhizobium meliloti is alternately capable of colonizing the soil as a free-living bacterium or establishing a chronic intracellular infection with its legume host for the purpose of nitrogen fixation. We previously identified the S. meliloti two-component sensor histidine kinase CbrA as playing an important role in regulating exopolysaccharide production, flagellar motility and symbiosis. Phylogenetic analysis of CbrA has highlighted its evolutionary relatedness to the Caulobacter crescentus sensor histidine kinases PleC and DivJ, which are involved in CtrA-dependent cell cycle regulation through the shared response regulator DivK. We therefore became interested in testing whether CbrA plays a role in regulating S. meliloti cell cycle processes. We find the loss of cbrA results in filamentous cell growth accompanied by cells that contain an aberrant genome complement, indicating CbrA plays a role in regulating cell division and possibly DNA segregation. S. meliloti DivK localizes to the old cell pole during distinct phases of the cell cycle in a phosphorylation-dependent manner. Loss of cbrA results in a significantly decreased rate of DivK polar localization when compared with the wild-type, suggesting CbrA helps regulate cell cycle processes by modulating DivK phosphorylation status as a kinase. Consistent with a presumptive decrease in DivK phosphorylation and activity, we also find the steady-state level of CtrA increased in cbrA mutants. Our data therefore demonstrate that CbrA contributes to free-living cell cycle regulation, which in light of its requirement for symbiosis, points to the potential importance of cell cycle regulation for establishing an effective host interaction.

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules.

Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

Role of specific quorum-sensing signals in the regulation of exopolysaccharide II production within Sinorhizobium meliloti spreading colonies.

BACKGROUND: Quorum sensing (QS) in Sinorhizobium meliloti involves at least half a dozen different N-acyl homoserine lactone (AHL) signals. These signals are produced by SinI, the sole AHL synthase in S. meliloti Rm8530. The sinI gene is regulated by two LuxR-type transcriptional regulators, SinR and ExpR. Mutations in sinI, sinR and expR abolish the production of exopolysaccharide II (EPS II). METHODOLOGY/PRINCIPAL FINDINGS: This study investigated a new type of coordinated surface spreading of Rm8530 that can be categorized as swarming. Motility assays on semi-solid surfaces revealed that both flagella and EPS II are required for this type of motility. The production of EPS II depends on AHLs produced by SinI. Of these AHLs, only C(16:1)- and 3-oxo-C(16:1)-homoserine lactones (HSLs) stimulated swarming in an ExpR-dependent manner. These two AHLs induced the strongest response in the wggR reporter fusions. WggR is a positive regulator of the EPS II biosynthesis gene expression. The levels of the wggR activation correlated with the extent of swarming. Furthermore, swarming of S. meliloti required the presence of the high molecular weight (HMW) fraction of EPS II. Within swarming colonies, a recombinase-based RIVET reporter in the wggR gene was resolved in 30% of the cells, indicating an enhanced regulation of EPS II production in the subpopulation of cells, which was sufficient to support swarming of the entire colony. CONCLUSIONS/SIGNIFICANCE: Swarming behavior of S. meliloti Rm8530 on semi-solid surfaces is found to be dependent on the functional QS regulatory cascades. Even though multiple AHL signals are produced by the bacterium, only two AHLs species, C(16:1)- and 3-oxo-C(16:1)-HSLs, affected swarming by up-regulating the expression of wggR. While EPS II is produced by Rm8530 as high and low molecular weight fractions, only the HMW EPS II facilitated initial stages of swarming, thus, suggesting a function for this polymer.

ExpR is not required for swarming but promotes sliding in Sinorhizobium meliloti.

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium Sinorhizobium meliloti Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of expR, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that S. meliloti can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional expR gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by S. meliloti is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.

Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis.

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

Biodegradation pathway and detoxification of the diazo dye Reactive Black 5 by Phanerochaete chrysosporium.

The in vivo biodegradation of the diazo dye Reactive Black 5 (RB5) by Phanerochaete chrysosporium immobilised on cubes of nylon sponge and on sunflower-seed shells (SS) in laboratory-scale bioreactors was investigated. The SS cultivation led to the best results with a decolouration percentage of 90.3% in 72 h for an initial RB5 concentration of 100 mg/L. It was found that the addition of 0.4 mM veratryl alcohol (VA) into the medium considerably increased the decolouration rate in SS cultivation. However, the addition of VA had no effect in the nylon cultivation. Thin layer chromatography (TLC) revealed that RB5 was transformed into one metabolite after 24 h. UV-vis spectroscopy and Fourier Transform Infrared (FT-IR) also confirmed the biodegradation of RB5. Toxicity of RB5 solutions before and after fungal treatment was assayed using Sinorhizobium meliloti as a sensitive soil microorganism. P. chrysosporium transformed the toxic dye RB5 into a non-toxic product.

Efficient and simple method for determination of suspendibility of bio-inoculant suspensions.

This study assessed the utilization of viscosity and zeta potential as a novel method to evaluate suspendibility of formulation of Sinorhizobium meliloti grown in starch industry wastewater for use as bio-inoculants. For this objective, sorbitol was used as a suspending agent at concentrations of 0 to 10% w/v. Model, based on multiple linear regression (with pH as dependant variable, and zeta potential, average particle size and sorbitol concentration as independent variables) demonstrated an important relation which was significant (p<0.001, R2=0.98). Sigmoid regression revealed a significant relation between zeta potential and suspendibility with p value=0.007 and R-squared=0.86, and between viscosity and suspendibility (p value<0.0001 and R squared=0.9823). Thus, these direct correlations established the lowering of measurement time from 12 h to 5 min.

Plant peptides govern terminal differentiation of bacteria in symbiosis.

Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation.

Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.