Competition in the Phaseolus vulgaris-Rhizobium symbiosis and the role of resident soil rhizobia in determining the outcomes of inoculation.

Background and Aims: Inoculation of legumes with effective N2-fixing rhizobia is a common practice to improve farming profitability and sustainability. To succeed, inoculant rhizobia must overcome competition for nodulation by resident soil rhizobia that fix N2 ineffectively. In Kenya, where Phaseolus vulgaris (common bean) is inoculated with highly effective Rhizobium tropici CIAT899 from Colombia, response to inoculation is low, possibly due to competition from ineffective resident soil rhizobia. Here, we evaluate the competitiveness of CIAT899 against diverse rhizobia isolated from cultivated Kenyan P. vulgaris. Methods: The ability of 28 Kenyan P. vulgaris strains to nodulate this host when co-inoculated with CIAT899 was assessed. Rhizosphere competence of a subset of strains and the ability of seed inoculated CIAT899 to nodulate P. vulgaris when sown into soil with pre-existing populations of rhizobia was analyzed. Results: Competitiveness varied widely, with only 27% of the test strains more competitive than CIAT899 at nodulating P. vulgaris. While competitiveness did not correlate with symbiotic effectiveness, five strains were competitive against CIAT899 and symbiotically effective. In contrast, rhizosphere competence strongly correlated with competitiveness. Soil rhizobia had a position-dependent numerical advantage, outcompeting seed-inoculated CIAT899 for nodulation of P. vulgaris, unless the resident strain was poorly competitive. Conclusion: Suboptimally effective rhizobia can outcompete CIAT899 for nodulation of P. vulgaris. If these strains are widespread in Kenyan soils, they may largely explain the poor response to inoculation. The five competitive and effective strains characterized here are candidates for inoculant development and may prove better adapted to Kenyan conditions than CIAT899.

The complete mitochondrial genome of the Australian ghost bat Macroderma gigas.

The Ghost bat Macroderma gigas is a monotypic bat species that is endemic to northern Australia and named on the basis of the large size of its partially conjoined ears. It is the only carnivorous bat found in Australia and its conservation status is currently listed as Vulnerable. Here, we describe the complete mitochondrial genome of M. gigas and compare it to other vertebrates. The M. gigas circularized mitogenome was 16,661 bp and contained 13 protein-coding genes, two rRNA genes, 22 tRNAs and a control region (D-loop) of 1228 bp. Phylogenetic analysis of available entire mitogenomes reveals that Macroderma gigas is most closely related to the Indian false vampire bat Megaderma lyra in the family Megadermatidae (false vampire bats).

The complete mitochondrial genome of the Australian Common Rock Rat, Zyzomys argurus.

The Common Rock Rat Zyzomys argurus is an abundant small- to medium-sized Murid rodent that is endemic to Australia. It is a nocturnal mammal with a mostly herbivorous diet. This species is native to the wet/dry tropics of Northern Australia and can be identified from other rock rats on the basis of its small size and its tail length (which is at least equivalent to its head-body length). Here, we describe the complete mitochondrial genome of Z. argurus and compare it to other Rodentia. The Z. argurus circular mitogenome was 16,261 bp and contained 13 protein-coding genes, two rRNA genes, 22 tRNAs and a control region (D-loop) of 859 bp. Phylogenetic analysis of selected, published sequenced mitogenomes reveal it is most closely related to the Lakeland Downs mouse Leggadina lakedownensis in the order Rodentia.

The complete mitochondrial genome of the vulnerable Australian crest-tailed mulgara (Dasycercus cristicauda).

In this announcement, we report the complete mitogenome of the vulnerable Crest-tailed Mulgara (Dasycercus cristicauda) (Krefft, 1867). The mitogenome was 17,085 bp in length and contained 13 protein-coding genes, two rRNA genes, 22 tRNAs and a 1583 bp variable control region (D-loop). The features of the D. cristicauda mitogenome are consistent with other vertebrate mitogenomes but, in contrast to other marsupials, appears to contain a functional tRNA-Lysine with a UUU anticodon. Phylogenetic analysis of available entire mitogenomes reveals it forms a cluster with other marsupials in the Dasyuromorphia order within the Australidelphian clade, being most closely related to the Northern Quoll and the Tasmanian Devil.

Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.

Discovery of Novel Plant Interaction Determinants from the Genomes of 163 Root Nodule Bacteria.

Root nodule bacteria (RNB) or "rhizobia" are a type of plant growth promoting bacteria, typified by their ability to fix nitrogen for their plant host, fixing nearly 65% of the nitrogen currently utilized in sustainable agricultural production of legume crops and pastures. In this study, we sequenced the genomes of 110 RNB from diverse hosts and biogeographical regions, and undertook a global exploration of all available RNB genera with the aim of identifying novel genetic determinants of symbiotic association and plant growth promotion. Specifically, we performed a subtractive comparative analysis with non-RNB genomes, employed relevant transcriptomic data, and leveraged phylogenetic distribution patterns and sequence signatures based on known precepts of symbiotic- and host-microbe interactions. A total of 184 protein families were delineated, including known factors for nodulation and nitrogen fixation, and candidates with previously unexplored functions, for which a role in host-interaction, -regulation, biocontrol, and more, could be posited. These analyses expand our knowledge of the RNB purview and provide novel targets for strain improvement in the ultimate quest to enhance plant productivity and agricultural sustainability.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

BACKGROUND AND AIMS: The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. METHODS: Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. KEY RESULTS: Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. CONCLUSIONS: Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with ≥ 96.1% sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFB(T), with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8% similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Microvirga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), WSM3557(T) and WSM3693(T) from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6(T) was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100% identical nodA sequences of WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), WSM3557(T) and WSM3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6(T) =LMG 26460(T) =HAMBI 3236(T)), Microvirga lotononidis sp. nov. (type strain WSM3557(T) =LMG 26455(T) =HAMBI 3237(T)) and Microvirga zambiensis sp. nov. (type strain WSM3693(T) =LMG 26454(T) =HAMBI 3238(T)).

Root nodule bacteria isolated from South African Lotononis bainesii, L. listii and L. solitudinis are species of Methylobacterium that are unable to utilize methanol.

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C(1) compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions.

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.

Probing for pH-regulated genes in Sinorhizobium medicae using transcriptional analysis.

The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis. Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021. Acid activated expression was detected for fixNO, kdpC, lpiA, and phrR and for genes encoding a putative lipoprotein, two ABC-transporter components, a putative DNA ligase and a MPA1-family protein. These findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S. medicae.

Probing for pH-regulated proteins in Sinorhizobium medicae using proteomic analysis.

To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media. Fifty pH-regulated proteins were identified; N-terminal sequences for 15 of these were obtained using the Edman degradation. Transient acid exposure downregulated GlnA and GlnK and upregulated a hypothetical protein. Continuing acid exposure downregulated ClpP, an ABC transporter, a hypothetical protein, a lipoprotein, the Trp-like repressor WrbA1 and upregulated DegP, fructose bisphosphate aldolase, GroES, malate dehydrogenase and two hypothetical proteins. These findings implicate proteolytic, chaperone and transport processes as key components of pH response in S. medicae.

Sinorhizobium medicae genes whose regulation involves the ActS and/or ActR signal transduction proteins.

ActS-ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global regulation in the alpha-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae. To do this, random gusA fusions were created in S. medicae to follow gene transcription in an actS chromosomal knockout mutant containing plasmid-borne actS. Plasmid borne actS was cured from the mutants and beta-glucuronidase (GUS) activity compared between the different genetic backgrounds. We detected actS-dependent regulation of the genes gst1 (detoxification), hyuA (hydantoin utilization) and fixN2 (microaerobic respiration). We show that ActR is involved in regulating cbbS (CO2 fixation), narB (nitrate assimilation) and required for low pH and microaerobic induction of the nitrogen fixation regulators fixK and nifA. In particular, we demonstrate that the transcriptional activation of fixN2 is regulated by ActR through FixK.

ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH-induced copper toxicity.

Two 'calcium-irreparable' acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily. Here, we show that both actP-knockout mutants show sensitivity to copper; omission of this heavy metal from low pH-buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actPgene. An actP-gusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH-dependent manner.Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal-responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional Inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses.

The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g. phosphate limitation, elevated temperature, high concentrations of sucrose or iron). A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3.5 kb BamHI fragment containing phrR from the mutant S. meliloti TG2-6 (actA::Tn5). DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced. The phrR open reading frame contiguous to actA appears to code for a 15.2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp. NGR234. All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage.

Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR.

A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp). At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.

An essential role for actA in acid tolerance of Rhizobium meliloti.

The actA gene, which is disrupted by Tn5 in the acid-sensitive mutant of Rhizobium meliloti TG2-6, was cloned and sequenced. It encodes a protein of 541 amino acids with a calculated molecular mass of 57,963 Da and an estimated pl of 9.0. The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Escherichia coli. Like the cutE mutant of E. coli, TG2-6 is sensitive to copper. The reconstructed wild-type actA gene complemented the low pH- and copper-sensitive phenotype of TG2-6. Studies with an actA-lacZ gene fusion showed that actA is constitutively expressed at pH 5.8 and 7.0. The actA gene appears to be chromosomal and is present in all seven strains of R. meliloti tested.