Direct measurement of the aerotactic response in a bacterial suspension.

Aerotaxis is the ability of motile cells to navigate toward oxygen. A key question is the dependence of the aerotactic velocity with the local oxygen concentration c. Here we combine simultaneous bacteria tracking and local oxygen concentration measurements using Ruthenium encapsulated in micelles to characterize the aerotactic response of Burkholderia contaminans, a motile bacterium ubiquitous in the environment. In our experiments, an oxygen gradient is produced by the bacterial respiration in a sealed glass capillary permeable to oxygen at one end, producing a bacterial band traveling toward the oxygen source. We compute the aerotactic response χ(c) both at the population scale, from the drift velocity in the bacterial band, and at the bacterial scale, from the angular modulation of the run times. Both methods are consistent with a power-law χ∝c^{-2}, in good agreement with existing models based on the biochemistry of bacterial membrane receptors.

Role of antimicrobial peptides in controlling symbiotic bacterial populations.

Covering: up to 2018 Antimicrobial peptides (AMPs) have been known for well over three decades as crucial mediators of the innate immune response in animals and plants, where they are involved in the killing of infecting microbes. However, AMPs have now also been found to be produced by eukaryotic hosts during symbiotic interactions with bacteria. These symbiotic AMPs target the symbionts and therefore have a more subtle biological role: not eliminating the microbial symbiont population but rather keeping it in check. The arsenal of AMPs and the symbionts' adaptations to resist them are in a careful balance, which contributes to the establishment of the host-microbe homeostasis. Although in many cases the biological roles of symbiotic AMPs remain elusive, for a number of symbiotic interactions, precise functions have been assigned or proposed to the AMPs, which are discussed here. The microbiota living on epithelia in animals, from the most primitive ones to the mammals, are challenged by a cocktail of AMPs that determine the specific composition of the bacterial community as well as its spatial organization. In the symbiosis of legume plants with nitrogen-fixing rhizobium bacteria, the host deploys an extremely large panel of AMPs - called nodule-specific cysteine-rich (NCR) peptides - that drive the bacteria into a terminally differentiated state and manipulate the symbiont physiology to maximize the benefit for the host. The NCR peptides are used as tools to enslave the bacterial symbionts, limiting their reproduction but keeping them metabolically active for nitrogen fixation. In the nutritional symbiotic interactions of insects and protists that have vertically transmitted bacterial symbionts with reduced genomes, symbiotic AMPs could facilitate the integration of the endosymbiont and host metabolism by favouring the flow of metabolites across the symbiont membrane through membrane permeabilization.

Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein.

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

Nod factor requirements for efficient stem and root nodulation of the tropical legume Sesbania rostrata.

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.

Molecular mechanisms of Nod factor diversity.

The rhizobia-legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.

Nod factors of Azorhizobium caulinodans strain ORS571 can be glycosylated with an arabinosyl group, a fucosyl group, or both.

In addition to the previously described arabinosylated Nod factors, Azorhizobium caulinodans can also produce fucosylated Nod factors and Nod factors that are both arabinosylated and fucosylated. The presence of a plasmid carrying extra copies of a subset of nod genes as well as bacterial growth conditions influence the relative proportion of carbamoylated, fucosylated, and arabinosylated Nod factors. By using a root hair formation assay, we demonstrate that the Nod factor glycosylations are important for biological activity on Sesbania rostrata roots.

The nodulation gene nolK of Azorhizobium caulinodans is involved in the formation of GDP-fucose from GDP-mannose.

The nolK gene of Azorhizobium caulinodans is essential for the incorporation of a fucosyl group in Nod factors. A NAD(P)-binding site is present in the NolK amino acid sequence and the gene is homologous to Escherichia coli genes, presumably involved in GDP-fucose synthesis. Protein extracts of A. caulinodans, overexpressing nolK, have an enzyme activity that synthesizes GDP-fucose from GDP-mannose. nolK most probably encodes a 4-reductase performing the last step in GDP-fucose synthesis. Wild-type A. caulinodans produces a population of fucosylated and non-fucosylated molecules but the nolK-overexpressing strain produces only fucosylated Nod factors. Thus, the production of activated fucosyl donors is a rate-limiting step in Nod factor fucosylation.

Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolK, nodZ as well as noeC and/or downstream genes.

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch. Nod factors produced by Tn5-insertion mutants in nodZ, noeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK. Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ. In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.

Role of nodl and nodJ in lipo-chitooligosaccharide secretion in Azorhizobium caulinodans and Escherichia coli.

Lipo-chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell-associated and secreted radioactively labelled Nod factors in wild-type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild-type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5 alpha. The strain DH5 alpha (pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans. nodl or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nodlJ-encoded transporter showed a specificity for more hydrophilic LCOs.

Biosynthesis of Azorhizobium caulinodans Nod factors. Study of the activity of the NodABCS proteins by expression of the genes in Escherichia coli.

By in vitro and in vivo studies with Escherichia coli expressing different combinations of the nodABCS genes of Azorhizobium caulinodans, Nod factor intermediates were identified and their structures determined using mass spectrometry. Substrate-product relationships were studied by time course experiments, and the Nod factor biosynthetic pathway was partially resolved. E. coli strains, harboring nodA and/or nodB, did not produce Nod metabolites, whereas the strain expressing nodC produced chitooligosaccharides. Thus, the first committed step was the production of the carbohydrate backbone. Bacitracin and tunicamycin did not affect this step, suggesting that undecaprenyl pyrophosphate-linked intermediates were not involved. The second step was the deacetylation of chitooligosaccharides by NodB since the E. coli strain expressing nodBC produced chitooligosaccharides, deacetylated at the non-reducing end and since the NodC products were precursors of the NodBC products. A strain expressing nodBCS produced N-methylated oligosaccharides, whereas a strain expressing nodCS produced unmethylated oligosaccharides. Time course experiments showed that methylation occurred after deacetylation. Thus, NodS acted after NodB. The NodBCS metabolites were partially converted to lipo-chitooligosaccharides when the nodABCS genes were expressed, showing that NodA was involved in the acylation and acted after NodS.

NodS is an S-adenosyl-L-methionine-dependent methyltransferase that methylates chitooligosaccharides deacetylated at the non-reducing end.

In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain-specific substitutions. In Azorhizobium caulinodans strain ORS571 these modifications are an O-arabinosyl group, an O-carbamoyl group, and an N-methyl group. Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors. Previously we have shown that NodS is an S-adenosyl-L-methionine (SAM)-binding protein, essential for the L-[3H-methyl]-methionine labelling of ORS571 Nod factors in vivo. Here, we present an in vitro assay showing that NodS from either A. caulinodans or Rhizobium species NGR234 methylates end-deacetylated chitooligosaccharides, using [3H-methyl]-SAM as a methyl donor. The enzymatic and SAM-binding activity were correlated with the nodS gene and localized within the soluble protein fraction. The A. caulinodans nodS gene was expressed in Escherichia coli and a glutathione-S-transferase-NodS fusion protein purified. This protein bound SAM and could methylate end-deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo-chitooligosaccharides. These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides.

The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase.

Nod factors are signal molecules produced by Azorhizobium, Bradyrhizobium, and Rhizobium species that trigger nodule formation in leguminous host plants. The backbone of Nod factors consists of a beta-1,4-N-acetylglucosamine oligosaccharide from which the N-acetyl group at the nonreducing end is replaced by a fatty acid. The nodABC gene products are necessary for backbone biosynthesis. By incubation of cell extracts from Azorhizobium caulinodans with radioactive uridine diphosphate-N-acetylglucosamine, Nod factor precursors were identified and characterized as beta-1,4-N-acetylglucosamine oligosaccharides. By analysis of different nod gene mutants and by expression of nodC in Escherichia coli, the N-acetylglucosaminyltransferase activity was ascribed to the NodC protein. The results suggest that the first step in biosynthesis of Nod factors is the assembly of the oligosaccharide chain.

Identification of nodSUIJ genes in Nod locus 1 of Azorhizobium caulinodans: evidence that nodS encodes a methyltransferase involved in Nod factor modification.

The Azorhizobium caulinodans strain ORS571 nodulation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to S-adenosylmethionine (SAM)-utilizing methyltransferases. A naringenin-inducible nodS-dependent protein of approximately 25 kDa could be cross-linked to radiolabelled SAM. By applying L-[methyl-3H]-methionine in vivo, Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis.

Three unusual modifications, a D-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571.

Azorhizobium caulinodans strain ORS571 is a symbiont of the tropical legume Sesbania rostrata. Upon nod gene induction with naringenin, strain ORS571 secretes into the culture medium Nod factors that morphologically change the host plant--in particular, deformed root hairs (Hai/Had) and meristematic foci are formed at the basis of lateral roots. The latter infrequently develop further into nodule-like structures. The azorhizobial Nod factors are chitin tetramers or pentamers, N-acylated at the nonreducing-end glucosamine with either vaccenic acid (C18:1) or stearic acid (C18:0). They, thus, resemble the previously described Nod factors from (brady)rhizobia. The backbone lipooligosaccharide is substituted with unusual modifications, presumably involved in host-specificity determination. There is a D-arabinose branch on the reducing end and an N-methyl and O-carbamoyl substitution on the nonreducing end of the oligosaccharide chain. The previously identified nod gene nolK may be involved in the synthesis of a D-arabinose derivative. The nodS gene product is probably responsible for the N-methylation of Nod factors.

Identification of a new inducible nodulation gene in Azorhizobium caulinodans.

The narrow host range bacterial strain Azorhizobium caulinodans ORS571 induces the formation of nitrogen-fixing nodules on the root and stem of the tropical legume Sesbania rostrata. Here, a new flavonoid-inducible locus of ORS571 is described, locus 4. The locus was identified and isolated via the occurrence of particular sequences, the gamma and delta elements. These elements are reiterated in the ORS571 genome, linked to symbiotic loci. Sequencing of locus 4 showed the presence of an open reading frame (ORF6) that is flanked downstream by a gamma element and upstream by a delta element. The gamma element is approximately 180 bp in size, and shows homology to the insertion element ISRm3, an insertion sequence belonging to a distinct class of IS elements. The delta element is about 300 bp in size and has homology with repeated sequences found in other Rhizobiaceae. The ORF6 gene product shows a low, but significant homology to the mouse mastocytoma antigen P35B (Szikora et al., EMBO J. 9: 1041-1050, 1990) and to a class of NAD/NADP-binding sugar epimerase/dehydrogenases (Pissowotzki et al., Mol. Gen. Genet. 231: 113-213, 1991). Immediately upstream from ORF6, a nod box-related sequence is present, the arrangement of which is fully consistent with a recently presented model for the nod box structure (Goethals et al., Proc. Natl. Acad. Sci. USA 89: 1646-1650, 1992). Insertional inactivation of ORF6 did not affect the nodulation and fixation performance on S. rostrata. However, on S. formosa roots the nodulation kinetics of such a mutant was clearly affected (about 5 days delay). We propose to call this new symbiotic gene nolK.