RESUMO
Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.
Assuntos
Ácidos Graxos/biossíntese , Lipídeo A/biossíntese , Mutação , Sinorhizobium meliloti/metabolismo , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fabaceae/microbiologia , Ácidos Graxos/genética , Lipídeo A/genética , Sinorhizobium meliloti/genética , Simbiose/fisiologiaRESUMO
BacA proteins play key roles in the chronic intracellular infections of Sinorhizobium meliloti, Brucella abortus and Mycobacterium tuberculosis within their respective hosts. S. meliloti, B. abortus and M. tuberculosis BacA-deficient mutants have increased resistance to the thiazole-modified peptide bleomycin. BacA has been previously hypothesized, but not experimentally verified, to be involved in bleomycin uptake. In this paper, we show that a BacA-dependent mechanism is the major route of bleomycin internalization in S. meliloti. We also determined that the B. abortus and S. meliloti BacA proteins are functional homologues and that the B. abortus BacA protein is involved in the uptake of both bleomycin and proline-rich peptides. Our findings also provide evidence that there is a second, BacA-independent minor mechanism for bleomycin internalization in S. meliloti. We determined that the BacA-dependent and -independent mechanisms of bleomycin uptake are energy-dependent, consistent with both mechanisms of bleomycin uptake involving transport systems.
Assuntos
Proteínas de Bactérias/metabolismo , Sinorhizobium meliloti/metabolismo , Tiazóis/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Bleomicina/metabolismo , Sinorhizobium meliloti/genéticaRESUMO
The inner membrane BacA protein is essential for the establishment of chronic intracellular infections by Sinorhizobium meliloti and Brucella abortus within plant and mammalian hosts, respectively. In their free-living state, S. meliloti and B. abortus mutants lacking BacA have reductions in their outer membrane lipid A very-long-chain fatty acid (VLCFA) contents and exhibit low-level resistance to the glycopeptide bleomycin in comparison to their respective parent strains. In this paper we investigate the hypothesis that BacA is involved in peptide uptake in S. meliloti. We determined that an S. meliloti DeltabacA mutant is completely resistant to a truncated form of the eukaryotic peptide Bac7, Bac7(1-16), and this phenotype appears to be independent of its lipid A alteration. Subsequently, we discovered that BacA and/or Escherichia coli SbmA is essential for fluorescently labeled Bac7(1-16) uptake in S. meliloti. Given that there are hundreds of root nodule-specific peptides within the legume host, our data suggest that BacA-mediated peptide uptake could play a central role in the chronic infection process of S. meliloti. However, since we determined that two symbiotically defective S. meliloti bacA site-directed mutants (with the Q193G and R389G mutations, respectively) with known reductions in their lipid A VLCFA contents are still capable of peptide uptake, these findings suggest that BacA-dependent peptide uptake cannot fully account for the essential role of BacA in the legume symbiosis. Further, they provide evidence that the BacA function that leads to the S. meliloti lipid A VLCFA modification plays a key role in the chronic infection of legumes.
Assuntos
Anti-Infecciosos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neutrófilos/química , Peptídeos Cíclicos/metabolismo , Sinorhizobium meliloti/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bovinos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana/métodos , Dados de Sequência Molecular , Mutação , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/metabolismoRESUMO
Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.