Hajime Kobayashi

How rhizobial symbionts invade plants: the Sinorhizobium – Medicago model

Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).

Molecular Determinants of a Symbiotic Chronic Infection

Rhizobial bacteria colonize legume roots for the purpose of biological nitrogen fixation. A complex series of events, coordinated by host and bacterial signal molecules, underlie the development of this symbiotic interaction. Rhizobia elicit de novo formation of a novel root organ within which they establish a chronic intracellular infection. Legumes permit rhizobia to invade these root tissues while exerting control over the infection process. Once rhizobia gain intracellular access to their host, legumes also strongly influence the process of bacterial differentiation that is required for nitrogen fixation. Even so, symbiotic rhizobia play an active role in promoting their goal of host invasion and chronic persistence by producing a variety of signal molecules that elicit changes in host gene expression. In particular, rhizobia appear to advocate for their access to the host by producing a variety of signal molecules capable of suppressing a general pathogen defense response.

Flavonoids, NodD1, NodD2, and Nod-Box NB15 Modulate Expression of the y4wEFG Locus That Is Required for Indole-3-Acetic Acid Synthesis in Rhizobium sp. strain NGR234

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.

Rhizobia utilize pathogen-like effector proteins during symbiosis

A type III protein secretion system (T3SS) is an important host range determinant for the infection of legumes by Rhizobium sp. NGR234. Although a functional T3SS can have either beneficial or detrimental effects on nodule formation, only the rhizobial‐specific positively acting effector proteins, NopL and NopP, have been characterized. NGR234 possesses three open reading frames potentially encoding homologues of effector proteins from pathogenic bacteria. NopJ, NopM and NopT are secreted by the T3SS of NGR234. All three can have negative effects on the interaction with legumes, but NopM and NopT also stimulate nodulation on certain plants. NopT belongs to a family of pathogenic effector proteases, typified by the avirulence protein, AvrPphB. The protease domain of NopT is required for its recognition and a subsequent strong inhibition in infection of Crotalaria juncea. In contrast, the negative effects of NopJ are relatively minor when compared with those induced by its Avr homologues. Thus NGR234 uses a mixture of rhizobial‐specific and pathogen‐derived effector proteins. Whereas some legumes recognize an effector as potentially pathogen‐derived, leading to a block in the infection process, others perceive both the negative‐ and positive‐acting effectors concomitantly. It is this equilibrium of effector action that leads to modulation of symbiotic development.

Essential Role for the BacA Protein in the Uptake of a Truncated Eukaryotic Peptide in Sinorhizobium meliloti

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.

BacA, an ABC Transporter Involved in Maintenance of Chronic Murine Infections with Mycobacterium tuberculosis

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

Flavonoid-Inducible Modifications to Rhamnan O Antigens Are Necessary for Rhizobium sp. Strain NGR234-Legume Symbioses

Rhizobium sp. strain NGR234 produces a flavonoid-inducible rhamnose-rich lipopolysaccharide (LPS) that is important for the nodulation of legumes. Many of the genes encoding the rhamnan part of the molecule lie between 87 degrees and 110 degrees of pNGR234a, the symbiotic plasmid of NGR234. Computational methods suggest that 5 of the 12 open reading frames (ORFs) within this arc are involved in synthesis (and subsequent polymerization) of L-rhamnose. Two others probably play roles in the transport of carbohydrates. To evaluate the function of these ORFs, we mutated a number of them and tested the ability of the mutants to nodulate a variety of legumes. At the same time, changes in the production of surface polysaccharides (particularly the rhamnan O antigen) were examined. Deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis. Mutation of y4gM (a member of the ATP-binding cassette transporter family) did not abolish production of the rhamnose-rich LPS but, unexpectedly, the mutant displayed a symbiotic phenotype very similar to that of strains unable to produce the rhamnan O antigen (NGRDeltarmlB-wbgA and NGROmegafixF). At least two flavonoid-inducible regulatory pathways are involved in synthesis of the rhamnan O antigen. Mutation of either pathway reduces rhamnan production. Coordination of rhamnan synthesis with rhizobial release from infection threads is thus part of the symbiotic interaction.

TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes

Infection of legumes by Rhizobium sp. NGR234 and subsequent development of nitrogen‐fixing nodules are dependent on the coordinated actions of Nod factors, proteins secreted by a type III secretion system (T3SS) and modifications to surface polysaccharides. The production of these signal molecules is dependent on plant flavonoids which trigger a regulatory cascade controlled by the transcriptional activators NodD1, NodD2, SyrM2 and TtsI. TtsI is known to control the genes responsible for T3SS function and synthesis of a symbiotically important rhamnose‐rich lipo‐polysaccharide, most probably by binding to cis elements termed tts boxes. Eleven tts boxes were identified in the promoter regions of target genes on the symbiotic plasmid of NGR234. Expression profiles of lacZ fusions to these tts boxes showed that they are part of a TtsI‐dependent regulon induced by plant‐derived flavonoids. TtsI was purified and demonstrated to bind directly to two of these tts boxes. DNase I footprinting revealed that TtsI occupied not only the tts box consensus sequence, but also upstream and downstream regions in a concentration‐dependent manner. Highly conserved bases of the consensus tts box were mutated and, although TtsI binding was still observed in vitro, gfp fusions were no longer transcribed in vivo. Random mutagenesis of a tts box‐containing promoter revealed more nucleotides critical for transcriptional activity outside of the consensus.

Role of BacA in Lipopolysaccharide Synthesis, Peptide Transport, and Nodulation by Rhizobium sp. Strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.