Sinorhizobium meliloti GR4 Produces Chromosomal- and pSymA-Encoded Type IVc Pili That Influence the Interaction with Alfalfa Plants.

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. Sinorhizobium meliloti contains two clusters of T4cP-related genes: flp-1 and flp-2, which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with flp-1 are involved in the competitive nodulation of alfalfa plants, but the role of flp-2 remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive S. meliloti GR4 strain and investigated the role of its flp clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double flp-cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA flp clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of flp-1 in the absence of flp-2 reduces the competitiveness for nodule occupation.

Biodegradation of Microtoxic Phenylpropanoids (Phenylpropanoic Acid and Ibuprofen) by Bacteria and the Relevance for Their Removal from Wastewater Treatment Plants.

The NSAID ibuprofen (2-(4-isobutylphenyl)propanoic acid) and the structurally related 3-phenylpropanoic acid (3PPA), are widely used pharmaceutical and personal care products (PPCPs) which enter municipal waste streams but whose relatively low rates of elimination by wastewater treatment plants (WWTPs) are leading to the contamination of aquatic resources. Here, we report the isolation of three bacterial strains from a municipal WWTP, which as a consortium are capable of mineralizing ibuprofen. These were identified as the Pseudomonas citronellolis species, termed RW422, RW423 and RW424, in which the first two of these isolates were shown to contain the catabolic ipf operon responsible for the first steps of ibuprofen mineralization. These ipf genes which are associated with plasmids could, experimentally, only be transferred between other Sphingomonadaceae species, such as from the ibuprofen degrading Sphingopyxis granuli RW412 to the dioxins degrading Rhizorhabdus wittichii RW1, generating RW421, whilst a transfer from the P. citronellolis isolates to R. wittichii RW1 was not observed. RW412 and its derivative, RW421, as well as the two-species consortium RW422/RW424, can also mineralize 3PPA. We show that IpfF can convert 3PPA to 3PPA-CoA; however, the growth of RW412 with 3PPA produces a major intermediate that was identified by NMR to be cinnamic acid. This and the identification of other minor products from 3PPA allows us to propose the major pathway used by RW412 to mineralize 3PPA. Altogether, the findings in this study highlight the importance of ipf genes, horizontal gene transfer, and alternative catabolic pathways in the bacterial populations of WWTPs to eliminate ibuprofen and 3PPA.

The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.

A novel and affordable bioaugmentation strategy with microbial extracts to accelerate the biodegradation of emerging contaminants in different media.

This study describes a new bioaugmentation alternative based on the application of aqueous aerated extracts from a biomixture acclimated with ibuprofen, diclofenac and triclosan. This bioaugmentation strategy was assayed in biopurification systems (BPS) and in contaminated aqueous solutions to accelerate the removal of these emerging contaminants. Sterilized extracts or extracts from the initial uncontaminated biomixture were used as controls. In BPS, the dissipation of 90% of diclofenac and triclosan required, respectively, 60 and 108 days less than in the controls. The metabolite methyl-triclosan was determined at levels 12 times lower than in controls. In the bioaugmented solutions, ibuprofen was almost completely eliminated (99%) in 21 days and its hydroxylated metabolites were also determined to be at lower levels than in the controls. The plasmidome of acclimated biomixtures and its extract appeared to maintain certain types of plasmids but degradation related genes became less evident. Several dominant OTUs found in the extract identified as Flavobacterium and Fluviicola of the phylum Bacteroidetes, Thermomicrobia (phylum Chloroflexi) and Nonomuraea (phylum Actinobacteria), may be responsible for the enhanced dissipation of these contaminants. This bioaugmentation strategy represents an advantageous tool to facilitate in situ bioaugmentation.

Rhizobial Volatiles: Potential New Players in the Complex Interkingdom Signaling With Legumes.

Bacteria release a wide range of volatile compounds that play important roles in intermicrobial and interkingdom communication. Volatile metabolites emitted by rhizobacteria can promote plant growth and increase plant resistance to both biotic and abiotic stresses. Rhizobia establish beneficial nitrogen-fixing symbiosis with legume plants in a process starting with a chemical dialog in the rhizosphere involving various diffusible compounds. Despite being one of the most studied plant-interacting microorganisms, very little is known about volatile compounds produced by rhizobia and their biological/ecological role. Evidence indicates that plants can perceive and respond to volatiles emitted by rhizobia. In this perspective, we present recent data that open the possibility that rhizobial volatile compounds have a role in symbiotic interactions with legumes and discuss future directions that could shed light onto this area of investigation.

Effluent decontamination by the ibuprofen-mineralizing strain, Sphingopyxis granuli RW412: Metabolic processes.

The high global consumption of ibuprofen and its limited elimination by wastewater treatment plants (WWTPs), has led to the contamination of aquatic systems by this common analgesic and its metabolites. The potentially negative environmental and public health effects of this emerging contaminant have raised concerns, driving the demand for treatment technologies. The implementation of bacteria which mineralize organic contaminants in biopurification systems used to decontaminate water or directly in processes in WWTPs, is a cheap and sustainable means for complete elimination before release into the environment. In this work, an ibuprofen-mineralizing bacterial strain isolated from sediments of the River Elbe was characterized and assayed to remediate different ibuprofen-polluted media. Strain RW412, which was identified as Sphingopyxis granuli, has a 4.48 Mb genome which includes plasmid sequences which harbor the ipf genes that encode the first steps of ibuprofen mineralization. Here, we confirm that these genes encode enzymes which initiate CoA ligation to ibuprofen, followed by aromatic ring activation by a dioxygenase and retroaldol cleavage to unequivocally produce 4-isobutylcatechol and propionyl-CoA which then undergo further degradation. In liquid mineral salts medium, the strain eliminated more than 2 mM ibuprofen within 74 h with a generation time of 16 h. Upon inoculation into biopurification systems, it eliminated repeated doses of ibuprofen within a few days. Furthermore, in these systems the presence of RW412 avoided the accumulation of ibuprofen metabolites. In ibuprofen-spiked effluent from a municipal WWTP, ibuprofen removal by this strain was 7 times faster than by the indigenous microbiota. These results suggest that this strain can persist and remain active under environmentally relevant conditions, and may be a useful innovation to eliminate this emerging contaminant from urban wastewater treatment systems.

Bacterial ecotoxicity and shifts in bacterial communities associated with the removal of ibuprofen, diclofenac and triclosan in biopurification systems.

The proliferation and possible adverse effects of emerging contaminants such as pharmaceutical and personal care products (PPCPs) in waters and the environment is a cause for increasing concern. We investigated the dissipation of three PPCPs: ibuprofen (IBP), diclofenac (DCF) and triclosan (TCS), separately and in mixtures, in the ppm range in biopurification system (BPS) microcosms, paying special attention to their effect on bacterial ecotoxicity, as well as bacterial community structure and composition. The results reveal that BPS microcosms efficiently dissipate IBP and DCF with 90% removed after 45 and 84 days of incubation, respectively. However, removal of TCS required a longer incubation period of 127 days for 90% removal. Furthermore, dissipation of the PPCPs was slower when a mixture of all three was applied to BPS microcosms. TCS had an initial negative effect on bacterial viability by a decrease of 34-43% as measured by live bacterial cell counts using LIVE/DEAD® microscopy; however, this effect was mitigated when the three PPCPs were present simultaneously. The bacterial communities in BPS microcosms were more affected by incubation time than by the PPCPs used. Nonetheless, the PPCPs differentially affected the composition and relative abundance of bacterial taxa. IBP and DCF initially increased bacterial diversity and richness, while exposure to TCS generally provoked an opposite effect without full recovery at the end of the incubation period. TCS, which negatively affected the relative abundance of Acidobacteria, Methylophilales, and Legionellales, had the largest impact on bacterial groups. Biomarker OTUs were identified in the BPS microcosms which were constrained to higher concentrations of the PPCPs and thus are likely to harbour degradation and/or detoxification mechanisms. This study reveals for the first time the effect of PPCPs on bacterial ecotoxicity and diversity in biopurification system microcosms and also facilitates the design of further applications of biomixtures to eliminate PPCPs.

Improvement of pesticide removal in contaminated media using aqueous extracts from contaminated biopurification systems.

Despite certain limitations, bioaugmentation enhances the efficiency of bioremediation systems. In this study, three aqueous extracts (APE, ACE and APE) from aged residual biomixtures in three biopurification systems (BPSs) exposed to pesticides at a pilot scale were found to improve pesticide removal. The addition of ACEs and AVEs to solutions containing the model compound diuron increased removal rates 6- and 17-fold, respectively, as compared to APEs. These extracts also increased the removal of the metabolite 3,4-dichloroaniline, while AVEs, in particular, were found to remove all pesticides within 9 days. Three metabolites less hazardous than 3,4-dichloroaniline were identified by SPME/GC/MS. AVEs, which also enhance linuron removal in liquid media, were found to increase diuron removal 6-fold in BPSs. We observed an increase in the relative abundance of taxa, such as Chloroflexi, Acidobacteria, Gemmatimonadetes, Firmicutes, Deinococcus-Thermus and especially Proteobacteria (10%), in AV biomixtures, as well as an enrichment of γ-proteobacteria and the actinobacterial genus Dokdonella in AVEs with respect to initial noncontaminated IV biomixture. We demonstrate that extracts containing a pollutant-acclimatized microbiome could be used as part of a bioaugmentation strategy to improve the functioning of on-farm BPSs and contaminated systems.

Draft Genome Sequences of Isolates from Sediments of the River Elbe That Are Highly Tolerant to Diclofenac.

Here, we report the genome sequences of one Achromobacter and four Pseudomonas strains isolated from sediments of the River Elbe which are highly tolerant toward the xenobiotic target compound diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) and emerging contaminant.

The Sycamore Maple Bacterial Culture Collection From a TNT Polluted Site Shows Novel Plant-Growth Promoting and Explosives Degrading Bacteria.

Military activities have worldwide introduced toxic explosives into the environment with considerable effects on soil and plant-associated microbiota. Fortunately, these microorganisms, and their collective metabolic activities, can be harnessed for site restoration via in situ phytoremediation. We characterized the bacterial communities inhabiting the bulk soil and rhizosphere of sycamore maple (Acer pseudoplatanus) in two chronically 2,4,6-trinitrotoluene (TNT) polluted soils. Three hundred strains were isolated, purified and characterized, a majority of which showed multiple plant growth promoting (PGP) traits. Several isolates showed high nitroreductase enzyme activity and concurrent TNT-transformation. A 12-member bacterial consortium, comprising selected TNT-detoxifying and rhizobacterial strains, significantly enhanced TNT removal from soil compared to non-inoculated plants, increased root and shoot weight, and the plants were less stressed than the un-inoculated plants as estimated by the responses of antioxidative enzymes. The sycamore maple tree (SYCAM) culture collection is a significant resource of plant-associated strains with multiple PGP and catalytic properties, available for further genetic and phenotypic discovery and use in field applications.

WHOP, a Genomic Region Associated With Woody Hosts in the Pseudomonas syringae Complex Contributes to the Virulence and Fitness of Pseudomonas savastanoi pv. savastanoi in Olive Plants.

Bacteria from the Pseudomonas syringae complex belonging to phylogroups 1 and 3 (PG1 and PG3, respectively) isolated from woody hosts share a genomic region herein referred to as WHOP (from woody host and Pseudomonas spp.), which is absent in strains infecting herbaceous organs. In this work, we show that this region is also encoded in P. syringae pv. actinidifoliorum (PG1) and six additional members of PG3, namely, Pseudomonas savastanoi pv. retacarpa, three P. syringae pathovars, Pseudomonas meliae, and Pseudomonas amygdali. Partial conservation of the WHOP occurs in only a few PG2 strains. In P. savastanoi pv. savastanoi NCPPB 3335, the WHOP region is organized into four operons and three independently transcribed genes. While the antABC and catBCA operons mediate the catabolism of anthranilate and catechol, respectively, the ipoABC operon confers oxygenase activity to aromatic compounds. The deletion of antABC, catBCA, or ipoABC in NCPPB 3335 caused reduced virulence in woody olive plants without affecting knot formation in nonwoody plants; catBCA, dhoAB, and PSA3335_3206 (encoding a putative aerotaxis receptor) were also required for the full fitness of this strain exclusively in woody olive plants. Overall, this study sheds light on the evolution and adaptation of bacteria from the P. syringae complex to woody hosts and highlights the enzymatic activities encoded within the WHOP region that are essential for this process.

Assessing Bacterial Diversity in the Rhizosphere of Thymus zygis Growing in the Sierra Nevada National Park (Spain) through Culture-Dependent and Independent Approaches.

Little is known of the bacterial communities associated with the rhizosphere of wild plant species found in natural settings. The rhizosphere bacterial community associated with wild thyme, Thymus zygis L., plants was analyzed using cultivation, the creation of a near-full length 16S rRNA gene clone library and 454 amplicon pyrosequencing. The bacterial community was dominated by Proteobacteria (mostly Alphaproteobacteria and Betaproteobacteria), Actinobacteria, Acidobacteria, and Gemmatimonadetes. Although each approach gave a different perspective of the bacterial community, all classes/subclasses detected in the clone library and the cultured bacteria could be found in the pyrosequencing datasets. However, an exception caused by inconclusive taxonomic identification as a consequence of the short read length of pyrotags together with the detection of singleton sequences which corresponded to bacterial strains cultivated from the same sample highlight limitations and considerations which should be taken into account when analysing and interpreting amplicon datasets. Amplicon pyrosequencing of replicate rhizosphere soil samples taken a year later permit the definition of the core microbiome associated with Thymus zygis plants. Abundant bacterial families and predicted functional profiles of the core microbiome suggest that the main drivers of the bacterial community in the Thymus zygis rhizosphere are related to the nutrients originating from the plant root and to their participation in biogeochemical cycles thereby creating an intricate relationship with this aromatic plant to allow for a feedback ecological benefit.

Draft Genome Sequence of Pseudomonas putida JLR11, a Facultative Anaerobic 2,4,6-Trinitrotoluene Biotransforming Bacterium.

We report the draft genome sequence of Pseudomonas putida JLR11, a facultative anaerobic bacterium that has been studied in detail for its capacity to use the explosive 2,4,6-trinitrotoluene (TNT) as a nitrogen source. The sequence confirms the mechanisms used by this versatile strain to reduce and assimilate nitrogen from TNT.

Bacterial diversity in the rhizosphere of maize and the surrounding carbonate-rich bulk soil.

Maize represents one of the main cultivar for food and energy and crop yields are influenced by soil physicochemical and climatic conditions. To study how maize plants influence soil microbes we have examined microbial communities that colonize maize plants grown in carbonate-rich soil (pH 8.5) using culture-independent, PCR-based methods. We observed a low proportion of unclassified bacteria in this soil whether it was planted or unplanted. Our results indicate that a higher complexity of the bacterial community is present in bulk soil with microbes from nine phyla, while in the rhizosphere microbes from only six phyla were found. The predominant microbes in bulk soil were bacteria of the phyla Acidobacteria, Bacteroidetes and Proteobacteria, while Gammaproteobacteria of the genera Pseudomonas and Lysobacter were the predominant in the rhizosphere. As Gammaproteobacteria respond chemotactically to exudates and are efficient in the utilization of plants exudate products, microbial communities associated to the rhizosphere seem to be plant-driven. It should be noted that Gammaproteobacteria made available inorganic nutrients to the plants favouring plant growth and then the benefit of the interaction is common.

Laboratory research aimed at closing the gaps in microbial bioremediation.

The industrial revolution, the first agricultural 'green revolution', and the development of antibiotics and therapeutic chemicals have brought significant and undeniable benefits to the human race. However, these advances demand high levels of energy, exploit natural resources and create large amounts of waste that creates an environmental burden for our planet. The pollution rate and character of many of the pollutants results in a rapid deterioration of the environment. Bioremediation functions to isolate and select microorganisms that operate under aerobic and anoxic conditions to remove these harmful pollutants. Current 'omics' technologies allow the exploitation of the catabolic potential of microbes without the need to cultivate them. Synthetic microbiology builds new catabolic pathways to remove recalcitrant pollutants from the environment.

Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain).

The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) × 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.

Transcriptome profiling of a Sinorhizobium meliloti fadD mutant reveals the role of rhizobactin 1021 biosynthesis and regulation genes in the control of swarming.

BACKGROUND: Swarming is a multicellular phenomenom characterized by the coordinated and rapid movement of bacteria across semisolid surfaces. In Sinorhizobium meliloti this type of motility has been described in a fadD mutant. To gain insights into the mechanisms underlying the process of swarming in rhizobia, we compared the transcriptome of a S. meliloti fadD mutant grown under swarming inducing conditions (semisolid medium) to those of cells grown under non-swarming conditions (broth and solid medium). RESULTS: More than a thousand genes were identified as differentially expressed in response to growth on agar surfaces including genes for several metabolic activities, iron uptake, chemotaxis, motility and stress-related genes. Under swarming-specific conditions, the most remarkable response was the up-regulation of iron-related genes. We demonstrate that the pSymA plasmid and specifically genes required for the biosynthesis of the siderophore rhizobactin 1021 are essential for swarming of a S. meliloti wild-type strain but not in a fadD mutant. Moreover, high iron conditions inhibit swarming of the wild-type strain but not in mutants lacking either the iron limitation response regulator RirA or FadD. CONCLUSIONS: The present work represents the first transcriptomic study of rhizobium growth on surfaces including swarming inducing conditions. The results have revealed major changes in the physiology of S. meliloti cells grown on a surface relative to liquid cultures. Moreover, analysis of genes responding to swarming inducing conditions led to the demonstration that iron and genes involved in rhizobactin 1021 synthesis play a role in the surface motility shown by S. meliloti which can be circumvented in a fadD mutant. This work opens a way to the identification of new traits and regulatory networks involved in swarming by rhizobia.

Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

Microorganisms and explosives: mechanisms of nitrogen release from TNT for use as an N-source for growth.

Unstable reduced derivatives of 2,4,6-trinitrotoluene (TNT) produced by microorganisms have been found to release nitrite by rearomatization and/or condensation. Here, we present further information regarding the novel mechanism of the condensation of reactive hydroxylaminodinitrotoluene and the Meisenheimer dihydride complex of TNT to produce two secondary diarylamine isomers. Using uniformly 15N-labeled (15N3) TNT, we show that the nitrite is being released by the condensation reaction and, also under environmental conditions, will originate from the microbiologically generated dihydride complex.

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants.

BACKGROUND: Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene. RESULTS: The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation. CONCLUSION: tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor, N-acetyl glucosamine reduces nod gene expression and nodulation efficiency when present at millimolar concentrations. A role for Tep1 in the efflux of Nod factor precursors could explain the phenotypes associated with tep1 inactivation.