Downregulation of miR-144 blocked the proliferation and invasion of nerve cells in Hirschsprung disease by regulating Transcription Factor AP 4 (TFAP4).

BACKGROUND: Hirschsprung's disease (HSCR) is characterized by a dysfunction of enteric neural crest cells (ENCCs) proliferation, migration and premature apoptosis during embryonic development, resulting in aganglionic colon. Our aim is to explore the role of miR-144 with its target gene Transcription Factor AP 4 (TFAP4) in nerve cells in HSCR. METHODS: The relative expression levels of miR-144 in HSCR colon samples were detected by quantitative real-time PCR (RT-qPCR). Western blot assays were conducted to investigate the TFAP4 protein expressing level. The interaction of miR-144 and TFAP4 was predicted with bioinformatics analysis and examined with luciferase reporter assays. Overexpression or knockdown of miR-144 and TFAP4 in 293T and SH-SY5Y cell lines was applied. Cell proliferation, migration and invasion were detected by CCK-8 assays, Transwell migration and invasion assays. Cell cycle and apoptosis was examined by flow cytometric analysis. RESULTS: Downregulation of miR-144 and upregulation of TFAP4 were shown in HSCR. Luciferase reporter assay indicated that miR-144 reduced luciferase activity in 293T and SH-SY5Y transfected with TFAP4-WT-3UTR luciferase reporter and confirmed TFAP4 was the downstream target gene of miR-144. Data showed that miR-144 promoted the cell proliferation, migration and invasion of 293T and SH-SY5Y, while TFAP4 blocked the cell proliferation, migration and invasion. TFAP4 overexpression reversed the miR-144-mediated cell proliferation, migration and invasion of 293T and SH-SY5Y. CONCLUSIONS: Downregulation of miR-144 blocked the cell proliferation and migration of nerve cells via targeting TFAP4 and contributed to the pathogenesis of HSCR. This provides an innovative and candidate target for treatment of HSCR.

The flavonoid luteolin suppresses infantile hemangioma by targeting FZD6 in the Wnt pathway.

Infantile hemangioma is the most common vascular tumor of childhood. It is characterized by clinical expansion of endothelial cells and promoted by angiogenic factors. Luteolin is a flavonoid compound that carries anti-cancer and anti-angiogenesis properties. The study aimed to investigate the effect of luteolin in treating infantile hemangioma. We first tested the effect of luteolin on cell proliferative potential and VEGFA expression in hemangioma-derived stem cells (HemSCs). We then examined the efficacy of systemic application of luteolin in a murine hemangioma model. We then identified the downstream factor regulated by luteolin in HemSCs and validated its causative relationship with knock-down method in both in vitro and in vivo models. We also investigated the protein expression change of this targeting factor in proliferating hemangiomas. Luteolin inhibited HemSC growth and suppressed VEGF-A expression in a dose-dependent manner. Luteolin inhibited microvessel formation and de novo vasculogenesis in the murine model. FZD6 was induced by luteolin and exerted the anti-angiogenesis effect in our tumor models. Lastly, FZD6 level was repressed in the clinical tissues of human proliferating hemangiomas. Luteolin is a promising new agent to treat infantile hemangioma. Targeting the Wnt pathway may represent a potential therapeutic strategic to inhibit angiogenesis in proliferating hemangiomas.

Pinitol suppresses TNF-α-induced chondrocyte senescence.

Osteoarthritis (OA) is a highly prevalent joint disorder that is tightly correlated with age. As the body ages, cell replication and function decline until homeostasis can no longer be maintained. This process involves cellular senescence as well as replicative senescence. Telomere length, cell cycle arrest, expression of p16 and p53, and the release of senescence-associated ß-galactosidase (SA-ß-Gal) are all markers of cell senescence. In OA joints, chondrocytes undergo cellular senescence prematurely, thereby ceasing to synthesize and maintain cartilage tissue. Upregulation of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), and oxidative stress induced by overproduction of reactive oxygen species (ROS) are key events in the pathogenesis of OA. In the present study, we investigated the effects of pinitol, a naturally occurring compound, on the effects of TNF-α on chondrocyte senescence and cell cycle arrest. We found that pinitol has a favorable safety profile in terms of cell viability. Pinitol significantly inhibited cellular senescence and cell cycle arrest in the G0/G1 phase induced by TNF-α. We also found that pinitol could inhibit TNF-α-induced increased telomerase activity and expression of p16 and p53. Importantly, we found that the effects of pinitol may be mediated through rescue of Nrf2 signaling, which is recognized as a key protective factor in OA. This finding was verified through a Nrf2 silencing experiment using Nrf2 siRNA. Together, our findings reveal the potential of pinitol as a safe therapeutic option for the prevention of OA-associated chondrocyte senescence and oxidative stress.

Long noncoding RNA LINC00342 promotes growth of infantile hemangioma by sponging miR-3619-5p from HDGF.

Infantile hemangiomas (IH) are a type of benign vascular neoplasm that may cause permanent scarring. Hemangioma-derived endothelial cells (HemECs) are commonly used as an in vitro model to study IH. Long noncoding RNA is a type of RNA transcript longer than 200 nucleotides that does not encode any protein. LINC00342 was discovered to regulate proliferation and apoptosis in nonsmall cell lung cancer. However, the role of LINC00342 in IH has never been reported before. Expressions of LINC00342 and miR-3619-5p were detected in proliferating versus normal skin tissues. Colony formation and Cell-Couting Kit 8 assays were carried out to study the effects on cell proliferation after knockdown and overexpression of LINC00342, respectively. Meanwhile caspase-3 activity and nucleosomal fragmentation assay were applied to detect cell apoptosis. Micro-RNA binding sites on LINC00342 and hepatoma-derived growth factor (HDGF) were predicted and confirmed via dual-luciferase reporter assay. Biotin RNA pulldown assay was used to verify the direct binding between RNA molecules. LINC00342 enhanced proliferation and inhibited apoptosis in HemECs. MiR-3619-5p targeted both LINC00342 and HDGF, where LINC00342 sponged miR-3619-5p and positively regulated HDGF. HDGF knockdown rescued the effects of LINC00342 on HemECs. The LINC00342-miR-3619-5p-HDGF signaling pathway could regulate cell proliferation and apoptosis in HemECs.NEW & NOTEWORTHY The role of LINC00342 in infantile hemangiomas has not yet been elucidated. This paper highlights the regulatory role of LINC00342 in cell proliferation and apoptosis in hemangioma-derived endothelial cells and the underlying molecular mechanisms. The findings would provide potential target for treatment of infantile hemangiomas.

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

The quorum sensing regulator CinR hierarchically regulates two other quorum sensing pathways in ligand-dependent and -independent fashions in Rhizobium etli.

UNLABELLED: Many rhizobial species use complex N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) systems to monitor their population density and regulate their symbiotic interactions with their plant hosts. There are at least three LuxRI-type regulatory systems in Rhizobium etli CFN42: CinRI, RaiRI, and TraRI. In this study, we show that CinI, RaiI, and TraI are responsible for synthesizing all AHLs under the tested conditions. The activation of these AHL synthase genes requires their corresponding LuxR-type counterparts. We further demonstrate that CinRI is at the top of the regulatory cascade that activates RaiRI and TraRI QS systems. Moreover, we discovered that CinR possesses a specific affinity to bind cinI promoter in the absence of its cognate AHL ligand, thereby activating cinI transcription. Addition of AHLs leads to improved binding to the cinI promoter and enhanced cinI expression. Furthermore, we found that compared to the wild type, the cinR mutation displayed reduced nodule formation, and cinR, raiR, and traI mutants show significantly lower levels of nitrogen fixation activity than the wild type. These results suggest that the complex QS regulatory systems in R. etli play an important role in its symbiosis with legume hosts. IMPORTANCE: Many bacteria use quorum sensing (QS) to monitor their cell densities and coordinately regulate a number of physiological functions. Rhizobia often have diverse and complex LuxR/LuxI-type quorum sensing systems that may be involved in symbiosis and N2 fixation. In this study, we identified three LuxR/LuxI-type QS systems in Rhizobium etli CFN42: CinRI, RaiRI, and TraRI. We established a complex network of regulation between these QS components and found that these QS systems played important roles in symbiosis processes.

Function of MsiR on canavanine-mediated repression in Mesorhizobium tianshanense.

Mesorhizobium tianshanense employs MsiA as canavanine exporter, which is upregulated by MsiR, to successfully form a symbiosis with the legume Glycyrrhiza uralensis. In this research, through gel-shift and bacterial two-hybrid examination, MsiR was found to spontaneously form dimers and bind to msiA promoter without additional canavanine. Six truncated forms of MsiR were constructed, and the conserved helix-turn-helix (HTH), substrate-binding, and surface-loop domains were found essential for MsiR functions. Random mutagenesis was used to study the functional sites of MsiR. Seven point mutants were selected, in which three mutants constitutively induced msiA expression without additional canavanine, two mutants partially changed substrate specificity, and the other two were almost null mutants. Results from the site mutation show that the functional subunits (HTH domain, dimerization interface domains, and C-terminal) are important in the conformation and induction ability of MsiR.

Flagellar-dependent motility in Mesorhizobium tianshanense is involved in the early stage of plant host interaction: study of an flgE mutant.

Bacterial motility is most likely a critical factor for rhizobium to chemotactically colonize on the root surface prior to infecting leguminous plant hosts. Several studies of the rhizobium flagellar filament have been reported, but little is known about the rhizobium flagellum hook. To investigate the roles of the hook protein in flagellum synthesis in Mesorhizobium tianshanense, the hook protein-encoding gene flgE of M. tianshanense was amplified by PCR and sequenced. Comparison of the deduced amino acid sequences revealed pronounced similarities in Domain 1 and lower similarities in Domain 2, which are supposed to be related to hook structure assembly and antigenic diversity, respectively. The level of transcription of flgE increased along with the cell growth and reached its maximum at the middle log phase. Disruption of the flgE gene caused a flagellar-less phenotype, thereby causing complete loss of swimming ability, modified nutrient-related swarming ability and biofilm formation. Moreover, the absence of flagellar caused decreased bacterial attachment on the root hair, suggesting that flagellar is involved in the early stage of symbiosis process.

Evaluation of quality and risk to human health of groundwater in a coastal coal-bearing graben basin, North China.

Water quality assessments are essential for ensuring human and ecosystem health. This study conducted a water quality assessment on a typical coastal coal-bearing graben basin. Suitability of groundwater quality of the basin for drinking and agricultural irrigation was assessed. Hazards posed to human health by groundwater nitrate were assessed using the objective combined weight water quality index, percent sodium, sodium adsorption ratio, and health risk assessment model. The results indicated groundwater of the basin to be weakly alkaline hard-fresh or hard-brackish, and mean values of pH, total dissolved solids, and total hardness of 7.6, 1464.5 mg/L, and 794.1 mg/L, respectively. The ranks of groundwater cations and anions by abundance were Ca2+>Na+>Mg2+>K+ and HCO3->NO3->Cl->SO42->F-, respectively. The main groundwater type was Cl-Ca, followed by HCO3-Ca. The results of the water quality evaluation indicated that most groundwater in the study area is of a medium quality (38%), followed by poor (33%), and extremely poor (26%). Groundwater quality gradually worsened from inland to the coast. Groundwater of the basin was generally suitable for agricultural irrigation. Groundwater nitrate posed a hazard to over 60% of the exposed population, with infants most at risk, followed by children, adult females, and adult males.

Insights for booster chlorination strategy based on DBPs control in a large-scale water supply system.

Based on a one-year field investigation of disinfection by-products (DBPs) in large scale water distribution system (LSWDS), the various characteristics of DBPs together with their correlation with booster chlorination were elaborated through ArcGIS model. Furthermore, the effects of booster chlorination on DBP formation were investigated through simulated experiments. Residual chlorine showed a strong relationship with occurrence of different DBPs in LSWDS, and the yield of DBPs increased significantly after booster chlorination. The simulated chlorination experiments showed that diminution of the ratio of primary to secondary (booster) chlorination dosage, and delaying the secondary chlorine addition reduced the generation of DBPs during water conveyance. The yield concentrations of THMs and HAAs obviously increased after booster chlorination. The correlation between HAAs and chlorine dosage is weaker in the field research than in the simulation experiment while THMs had a positive correlation with the chlorine addition in both field research and simulation experiment.

The effects of vaccination on the disease severity and factors for viral clearance and hospitalization in Omicron-infected patients: A retrospective observational cohort study from recent regional outbreaks in China.

Object: This study attempted to explore the effects of vaccination on disease severity and the factors for viral clearance and hospitalization in omicron-infected patients. Methods: The clinical manifestations of 3,265 Omicron-infected patients (BA.2 lineage variant; the Omicron group) were compared with those of 226 Delta-infected patients (the Delta group). A Multi-class logistic regression model was employed to analyze the impacts of vaccination doses and intervals on disease severity; a logistic regression model to evaluate the risk factors for hospitalization; R 4.1.2 data analysis to investigate the factors for time for nucleic acid negativization (NAN). Results: Compared with the Delta group, the Omicron group reported a fast transmission, mild symptoms, and lower severity incidence, and a significant inverse correlation of vaccination dose with clinical severity (OR: 0.803, 95%CI: 0.742-0.868, p<0.001). Of the 7 or 5 categories of vaccination status, the risk of severity significantly decreased only at ≥21 days after three doses (OR: 0.618, 95% CI: 0.475-0.803, p<0.001; OR: 0.627, 95% CI: 0.482-0.815, p<0.001, respectively). The Omicron group also reported underlying illness as an independent factor for hospitalization, sore throat as a protective factor, and much shorter time for NAN [15 (12,19) vs. 16 (12,22), p<0.05]. NAN was associated positively with age, female gender, fever, cough, and disease severity, but negatively with vaccination doses. Conclusion: Booster vaccination should be advocated for COVID-19 pandemic-related control and prevention policies and adequate precautions should be taken for patients with underlying conditions.

Host legume-exuded antimetabolites optimize the symbiotic rhizosphere.

Rhizobia form symbiotic nodules on host legumes and fix nitrogen for their hosts in exchange for nutrients. In order to establish this mutually beneficial relationship, rhizobia must compete with other soil bacteria in the host legume rhizosphere to colonize plant roots efficiently. A promoter-trap transposon screen in Mesorhizobium tianshanense, a Rhizobium that forms nodules on licorice (Glycyrrhiza uralensis) plants revealed that the expression of msiA, which encodes a putative exporter protein belonging to the LysE family of translocators, is activated by both legume exudates and MsiR, a LysR family transcriptional regulator. Chemical analysis suggests that the msiA-inducing signal in exudates is canavanine, an anti-metabolite present in the seeds and exudates of a variety of legume plants. We show that MsiA serves as a canavanine exporter that is indispensable for canavanine resistance in M. tianshanense. We also show that the expression of MsiA homologues in other rhizobial species is induced by canavanine and is critical for canavanine resistance. Furthermore, rhizobial canavanine resistance is important for root hair adherence as well as for survival in a canavanine-producing legume rhizosphere. Together, these data suggest that host legumes may exude specific antimetabolites into their surroundings to optimize the bacterial population in order to have successful symbiotic events with rhizobia.

Complex quorum-sensing regulatory systems regulate bacterial growth and symbiotic nodulation in Mesorhizobium tianshanense.

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process.

[Identification and functional characterization of genes induced by seed exudates in Azorhizobium caulinodans ORS571].

OBJECTIVE: To identify genes induced by plant seed exudates in Azorhizobium caulinodans ORS571. METHODS: Using promoterless kanamycin resistance gene (Km(r)) on transposon as reporter gene and seed exudates as inducers, we screened genes of interest from transposon insertion mutants libraries. We streaked mutants on TY solid medium with Km, and another with Km and seed exudates correspondingly. If Km(r) is inserted into a gene that can be induced by plant signals, Km(r) will possibly express at the same time. Thus, mutants were selected that can grow on medium with Km and exudates, rather than on medium with Km. RESULTS: We identified a lysE family gene named asiE in strain Azc0 that can be induced by seed exudates and further analysis indicated that the inducing substance is canavanine (CAN). lacZ transcriptional fusion of asiE confirmed that its expression increased by ten-fold or so under the induction of CAN. Besides, lysE gene in four different species of Rhizobia can be induced by CAN. lysE mutants are all sensitive to CAN treatment whereas wild type are resistant. CONCLUSION: The existence of LysE can make rhizobia better survived in the rhizosphere and may play an important role in early stage of interaction between rhizobia and host plant.

Internal Curing Effect and Compressive Strength Calculation of Recycled Clay Brick Aggregate Concrete.

In order to investigate the internal curing effect of recycled brick aggregate (RBA) in recycled aggregate concrete (RAC) and calculate its contribution to the final compressive strength, two RAC groups with different recycled aggregates and 6 replacement ratios (r) under 4 curing ages were tested. Results show that the compressive strengths of RACI and RACII decrease steadily with the increase of r when below 40%, and that there is a significant drop once the r is higher than 60%. The internal curing effect for RAC with a low RBA ratio is mainly reflected during the curing age of 14-21 days, while for RAC with a high RBA ratio, this internal curing effect appears earlier, during 7-14 days, and becomes very obvious after 14 days. In addition, the actual tested compressive strength of RAC replaced by 100% RBA exceeds around 40% of the expected compressive strength at the age of 28 days. When the age of RAC entirely with RBA is 28 days, the compressive strength caused by the internal curing effect accounts for around 28% of the actual tested compressive strength. The most appropriate r of RBA for RAC production is between 40% to 60%. Finally, the equations for calculating the compressive strength of RAC are presented considering the curing ages, the replacement ratios and the internal curing effect of RBA. Further, a unified equation is suggested for convenience in calculation.

[Recombinant expression of an autoinducer synthase of Sinorhizobium sp.1128 in Escherichia coli].

OBJECTIVE: To explore the roles of quorum sensing system in establishing symbiosis between bacterium Sinorhizobium sp.1128 and its plant host Melilotus suaveolens Ledeb. METHODS: According to homologous analysis, we designed primers to amplify the autoinducer synthase encoding genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences. The autoinducer synthase encoding genes were cloned into the expression vector of pYC12 and expressed in E. coli DH5alpha. Thin-layer chromatography (TLC) assay was used to study their roles in autoinducer production. A duplicated inactivation of the gene was used to explore its function in plant nodulation. RESULTS: Homologous analysis showed that at least three annotated acylated homoserine lactone (AHL) synthase genes existed in Sinorhizobium medicae WSM419 genome. We cloned these three autoinducer synthase genes in Sinorhizobium sp.1128. One of these genes named traI2 was over expressed in E. coli DH5alpha. At least two different AHLs were produced by the recombinant strain. Disruption of traI2 reduced both the autoinducers (AI) activities and AHL production by TLC detection. Furthermore, the complementation of traI2 reverted the phenotype of AI activities. These findings demonstrate that traI2 was responsible for AI synthesis in Sinorhizobium sp.1128. More important, the traI2 deficient strains were defective in nodule formation on their host plant. CONCLUSION: The quorum sensing circuits in Sinorhizobium sp.1128 may play an important role in symbiosis between plant and bacterium.

[Screening for autoinducer synthase gene in Sinorhizobium meliloti and analysis of the autoinducer produced by recombinant expression in Escherichia coli].

The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5alpha, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.

[Study on AHL-like quorum signals in Mesorhizobium huakuii].

Quorum sensing is a mechanism used by single-cell bacteria to monitor their population density and control a variety of physiological functions in a cell-density-dependent manner. An ultrasensitive quorum signal acyl-homoserine lactone (AHL) detecting Agrobacterium tumefaciens strain, which strongly overproduces quorum-sensing regulator TraR and was proved to be able to detect AHLs at nano-molar concentration, was used to detect the production of AHLs in three Mesorhizobium huakuii strains. We showed that AHLs were produced in these strains with some variations. We also found that elevating of medium pH during cell growth greatly affected the stability of AHL signals. This study paved the way of further studying the relationship between quorum sensing and symbiotic nitrogen fixation of the M. huakuii strains.

[A pedigree with myotonic dystrophy: non-CTG, non-CCTG repeat expansion].

OBJECTIVE: Two genetic loci are associated with the myotonic dystrophy (DM) phenotype: DM1 DMPK on chromosome 19, and DM2 ZNF9 on chromosome 3. The aim of this study was to investigate the molecular genetics of a pedigree with DM. METHODS: In twenty-six individuals from a family with DM, the CTG repeats in DMPK and CCTG repeats in ZNF9were evaluated genetically, using Long Expand trade mark Template polymerase chain reaction (PCR), Southern blotting and genomic scanning. RESULTS: The numbers of CTG and CCTG repeat were all in normal range. There was no significant difference between the CTG repeat size in DMPK gene and that 4 years later from the same individual. The Lod score values with short tandem repeats STR markers chosen in 19q and 3q were all smaller than 1, which suggested that no STR marker was linked with this DM family. CONCLUSION: There might be some other mutant in this DM pedigree. Further study should be done to find the genetic basis of this pedigree.

Applying reversible mutations of nodulation and nitrogen-fixation genes to study social cheating in Rhizobium etli-legume interaction.

Mutualisms are common in nature, though these symbioses can be quite permeable to cheaters in situations where one individual parasitizes the other by discontinuing cooperation yet still exploits the benefits of the partnership. In the Rhizobium-legume system, there are two separate contexts, namely nodulation and nitrogen fixation processes, by which resident Rhizobium individuals can benefit by cheating. Here, we constructed reversible and irreversible mutations in key nodulation and nitrogen-fixation pathways of Rhizobium etli and compared their interaction with plant hosts Phaseolus vulgaris to that of wild type. We show that R. etli reversible mutants deficient in nodulation factor production are capable of intra-specific cheating, wherein mutants exploit other Rhizobium individuals capable of producing these factors. Similarly, we show that R. etli mutants are also capable of cheating inter-specifically, colonizing the host legume yet contributing nothing to the partnership in terms of nitrogen fixation. Our findings indicate that cheating is possible in both of these frameworks, seemingly without damaging the stability of the mutualism itself. These results may potentially help explain observations suggesting that legume plants are commonly infected by multiple bacterial lineages during the nodulation process.