Connected Dominating Set (CDS) has been proposed as the virtual backbone to alleviate the broadca... more Connected Dominating Set (CDS) has been proposed as the virtual backbone to alleviate the broadcasting storm in wireless ad hoc networks. Most recent research has extensively focused on the construction of 1-Connected 1-Dominating Set (1-CDS) in homogeneous networks. However, the nodes in the CDS need to carry other node’s traffic and nodes in wireless networks are subject to failure. Therefore, it is desirable to construct a fault tolerant CDS. In this paper, we study a general fault tolerant CDS problem, called kk-Connected mm-Dominating Set (kk-mm-CDS), in heterogeneous networks. We first present two approximation algorithms for 1-mm-CDS and kk-kk-CDS problems. Using disk graphs to model heterogeneous networks, we show that our algorithms have a constant approximation ratio. Based on these two algorithms, we further develop a general algorithm for kk-mm-CDS. We also provide an interesting analysis for a special case of kk-mm-CDS, where k=m+1k=m+1.
Broadcast scheduling is a fundamental problem in wireless ad hoc networks. The objective of a bro... more Broadcast scheduling is a fundamental problem in wireless ad hoc networks. The objective of a broadcast schedule is to deliver a message from a given source to all other nodes in a minimum amount of time. At the same time, in order for the broadcast to proceed as predicted in the schedule, it must not contain parallel transmissions which can be conflicting based on the collision and interference parameters in the wireless network. Most existing work on this problem use a limited network model which accounts only for conflicts occurring inside the transmission ranges of the nodes. The broadcast schedules produced by these algorithms are likely to experience unpredictable delays when deployed in the network. This is because they do not take into consideration other important sources of conflict in parallel transmissions, namely the interference range and the carrier sensing range. In this paper we develop a conflict-aware network model, which uses these parameters to increase the probability of scheduling conflict-free transmissions, and thereby improve the reliability of the broadcast schedule. We present and prove correctness of a constant approximation algorithm for minimum-latency broadcast scheduling under this network model. We also present a greedy heuristic algorithm for the same problem. Experimental results are provided to evaluate the performance of our algorithms. In addition, the algorithms are analyzed to justify their performance trends.
An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by TnS mutagenesis, fail... more An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by TnS mutagenesis, fails to grow below pH 6 9 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking TnS in Murdoch, Western Australia 61 50, Australia TG5-46 contains two open reading frames, ORFl (designated acts) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into acts in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (Acts-) and TG5-46 (ActR-), while fragments containing only actR or acts complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complemented not only TG5-46 but also RT295. Cloning DNA upstream from actR and acts into a broad-host-range lac2 expression vector and measuring bgalactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria. 1693 0002-0535 0 1996 SGM actR/S : a two-component sensor-regulator system
• Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, compleme... more • Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021–M. truncatula symbiosis at fixing N2 was evaluated.• N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed.• Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells.• The Sm1021–M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021–M. truncatula symbiosis at fixing N2 was evaluated.N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed.Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells.The Sm1021–M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.
Journal of Molecular Microbiology and Biotechnology, 2004
The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivit... more 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.
The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by t... more The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by the nodulation of legumes by rhizobia that are ineffective or poorly effective in N2 fixation. This study investigated the development of rhizobial diversity for the pasture legume Biserrula pelecinus L., 6 years after its introduction, and inoculation with Mesorhizobium ciceri bv. biserrulae strain WSM1271, to Western Australia. Molecular fingerprinting of 88 nodule isolates indicated seven were distinctive. Two of these were ineffective while five were poorly effective in N2 fixation on B. pelecinus. Three novel isolates had wider host ranges for nodulation than WSM1271, and four had distinct carbon utilization patterns. Novel isolates were identified as Mesorhizobium sp. using 16S rRNA, dnaK and GSII phylogenies. In a second study, a large number of nodules were collected from commercially grown B. pelecinus from a broader geographical area. These plants were originally inoculated with M. c bv. biserrulae WSM1497 5–6 years prior to isolation of strains for this study. Nearly 50% of isolates from these nodules had distinct molecular fingerprints. At two sites diverse strains dominated nodule occupancy indicating recently evolved strains are highly competitive. All isolates tested were less effective and six were ineffective in N2 fixation. Twelve randomly selected diverse isolates clustered together, based on dnaK sequences, within Mesorhizobium and distantly to M. c bv. biserrulae. All 12 had identical sequences for the symbiosis island insertion region with WSM1497. This study shows the rapid evolution of competitive, yet suboptimal strains for N2 fixation on B. pelecinus following the lateral transfer of a symbiosis island from inoculants to other soil bacteria.
The actA gene, which is disrupted by TnS in the acid-sensitive mutant of Rhizobium meliloti TG2-6... more The actA gene, which is disrupted by TnS 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 57963 Da and an estimated pl of 99. The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Zscherichia coli. Like the cut€ 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-lacZgene fusion showed that actA is constitutively expressed at pH 58 and 7.0. The actA gene appears to be chromosomal and is present in all seven strains of R. meliloti tested.
Office of the Cassettes have been developed that contain an antibiotic resistance marker with and... more Office of the Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (PtaC) and the trpA terminator and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )
Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA ... more Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.
The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulg... more The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulgaris cultivated in Tunisian soils. This strain was selected on the basis of its high symbiotic effectiveness in laboratory conditions. In order to assess its ability to compete indigenous rhizobia, this strain was labelled with gusA gene. Conservation of initial effectiveness and competitiveness by transconjugants was tested. A transconjugant was introduced in three soil-core microcosms originating from different geographical and agronomic regions. Nodulation monitoring showed that the labelled transconjugant was able to occupy more than 90% of nodules at 30 days after inoculation. The nodule occupancy by the introduced strain was high even in the soil sample of Mateur which showed an MPN value of 103 rhizobia/g of dry soil. A significant improvement of plant productivity by inoculation was observed with the three soil samples in green house. Field inoculation with the parental strain showed a significant increase in nodule number, pod number and seed dry weight. The improvement of plant productivity in green house or in field conditions was equal or better than nitrogen fertilisation.
A mildly acid-sensitive mutant o f Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced co... more A mildly acid-sensitive mutant o f 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 o f T n 5 and the observed mucoid phenotype, acid sensitivity and TnS-induced kanamycin resistance were 100 O/ O co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid o f a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRl 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 TnS in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti R m l 0 2 l (71-3 O/ O identity over 892 bp). A t the protein level there was 70% identity and 9303% similarity over 267 amino acids with the ExoR protein of R. meliloti RmlO21.
Two ‘calcium-irreparable’ acid-sensitive mutants were identified after mutagenizing Rhizobium leg... more 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 actP gene. 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.
Journal of Molecular Microbiology and Biotechnology, 2004
To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we ha... more 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.
Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse r... more Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.
The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to... more 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.
Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range ... more Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome sequence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a feature unique to this medic microsymbiont. Editorial note -Readers are advised that in Opinion 84 the Judicial Commission of the International Committee on Systematics of Prokaryotes ruled that the genus name Ensifer Casida 1982 has priority over Sinorhizobium Chen et al. 1988 and the names are synonyms [1]. It was further concluded that the transfer of members of the genus Sinorhizobium to the genus Ensifer, as proposed by Young [2] would not cause confusion.
The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulg... more The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulgaris cultivated in Tunisian soils. This strain was selected on the basis of its high symbiotic effectiveness in laboratory conditions. In order to assess its ability to compete indigenous rhizobia, this strain was labelled with gusA gene. Conservation of initial effectiveness and competitiveness by transconjugants was tested. A transconjugant was introduced in three soil-core microcosms originating from different geographical and agronomic regions. Nodulation monitoring showed that the labelled transconjugant was able to occupy more than 90% of nodules at 30 days after inoculation. The nodule occupancy by the introduced strain was high even in the soil sample of Mateur which showed an MPN value of 10 3 rhizobia/g of dry soil. A significant improvement of plant productivity by inoculation was observed with the three soil samples in green house. Field inoculation with the parental strain showed a significant increase in nodule number, pod number and seed dry weight. The improvement of plant productivity in green house or in field conditions was equal or better than nitrogen fertilisation.
Rhizob ium le guminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-neg ati... more Rhizob ium le guminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-neg ative, non-spore-forming rod that was isolated from an ineffective root nodule recovered from the roots of the annual clover Trifolium rueppellianum Fresen g rowing in Ethiopia. WSM2012 has a na rrow, specialized host rang e for N 2 -fixation. Here we describe the features of R. leguminosarum bv. trifolii strain WSM2012, tog ether with genome sequence information and annotation. The 7,180,565 bp hig h-quality-draft genome is arrang ed into 6 scaffolds of 68 contig s, contains 7,080 protein-coding g enes and 86 RNA-only encoding genes, and is one of 20 rhizobial g enomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Prog ram.
Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse r... more Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.
Connected Dominating Set (CDS) has been proposed as the virtual backbone to alleviate the broadca... more Connected Dominating Set (CDS) has been proposed as the virtual backbone to alleviate the broadcasting storm in wireless ad hoc networks. Most recent research has extensively focused on the construction of 1-Connected 1-Dominating Set (1-CDS) in homogeneous networks. However, the nodes in the CDS need to carry other node’s traffic and nodes in wireless networks are subject to failure. Therefore, it is desirable to construct a fault tolerant CDS. In this paper, we study a general fault tolerant CDS problem, called kk-Connected mm-Dominating Set (kk-mm-CDS), in heterogeneous networks. We first present two approximation algorithms for 1-mm-CDS and kk-kk-CDS problems. Using disk graphs to model heterogeneous networks, we show that our algorithms have a constant approximation ratio. Based on these two algorithms, we further develop a general algorithm for kk-mm-CDS. We also provide an interesting analysis for a special case of kk-mm-CDS, where k=m+1k=m+1.
Broadcast scheduling is a fundamental problem in wireless ad hoc networks. The objective of a bro... more Broadcast scheduling is a fundamental problem in wireless ad hoc networks. The objective of a broadcast schedule is to deliver a message from a given source to all other nodes in a minimum amount of time. At the same time, in order for the broadcast to proceed as predicted in the schedule, it must not contain parallel transmissions which can be conflicting based on the collision and interference parameters in the wireless network. Most existing work on this problem use a limited network model which accounts only for conflicts occurring inside the transmission ranges of the nodes. The broadcast schedules produced by these algorithms are likely to experience unpredictable delays when deployed in the network. This is because they do not take into consideration other important sources of conflict in parallel transmissions, namely the interference range and the carrier sensing range. In this paper we develop a conflict-aware network model, which uses these parameters to increase the probability of scheduling conflict-free transmissions, and thereby improve the reliability of the broadcast schedule. We present and prove correctness of a constant approximation algorithm for minimum-latency broadcast scheduling under this network model. We also present a greedy heuristic algorithm for the same problem. Experimental results are provided to evaluate the performance of our algorithms. In addition, the algorithms are analyzed to justify their performance trends.
An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by TnS mutagenesis, fail... more An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by TnS mutagenesis, fails to grow below pH 6 9 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking TnS in Murdoch, Western Australia 61 50, Australia TG5-46 contains two open reading frames, ORFl (designated acts) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into acts in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (Acts-) and TG5-46 (ActR-), while fragments containing only actR or acts complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complemented not only TG5-46 but also RT295. Cloning DNA upstream from actR and acts into a broad-host-range lac2 expression vector and measuring bgalactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria. 1693 0002-0535 0 1996 SGM actR/S : a two-component sensor-regulator system
• Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, compleme... more • Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021–M. truncatula symbiosis at fixing N2 was evaluated.• N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed.• Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells.• The Sm1021–M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021–M. truncatula symbiosis at fixing N2 was evaluated.N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed.Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells.The Sm1021–M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.
Journal of Molecular Microbiology and Biotechnology, 2004
The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivit... more 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.
The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by t... more The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by the nodulation of legumes by rhizobia that are ineffective or poorly effective in N2 fixation. This study investigated the development of rhizobial diversity for the pasture legume Biserrula pelecinus L., 6 years after its introduction, and inoculation with Mesorhizobium ciceri bv. biserrulae strain WSM1271, to Western Australia. Molecular fingerprinting of 88 nodule isolates indicated seven were distinctive. Two of these were ineffective while five were poorly effective in N2 fixation on B. pelecinus. Three novel isolates had wider host ranges for nodulation than WSM1271, and four had distinct carbon utilization patterns. Novel isolates were identified as Mesorhizobium sp. using 16S rRNA, dnaK and GSII phylogenies. In a second study, a large number of nodules were collected from commercially grown B. pelecinus from a broader geographical area. These plants were originally inoculated with M. c bv. biserrulae WSM1497 5–6 years prior to isolation of strains for this study. Nearly 50% of isolates from these nodules had distinct molecular fingerprints. At two sites diverse strains dominated nodule occupancy indicating recently evolved strains are highly competitive. All isolates tested were less effective and six were ineffective in N2 fixation. Twelve randomly selected diverse isolates clustered together, based on dnaK sequences, within Mesorhizobium and distantly to M. c bv. biserrulae. All 12 had identical sequences for the symbiosis island insertion region with WSM1497. This study shows the rapid evolution of competitive, yet suboptimal strains for N2 fixation on B. pelecinus following the lateral transfer of a symbiosis island from inoculants to other soil bacteria.
The actA gene, which is disrupted by TnS in the acid-sensitive mutant of Rhizobium meliloti TG2-6... more The actA gene, which is disrupted by TnS 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 57963 Da and an estimated pl of 99. The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Zscherichia coli. Like the cut€ 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-lacZgene fusion showed that actA is constitutively expressed at pH 58 and 7.0. The actA gene appears to be chromosomal and is present in all seven strains of R. meliloti tested.
Office of the Cassettes have been developed that contain an antibiotic resistance marker with and... more Office of the Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (PtaC) and the trpA terminator and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )
Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA ... more Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.
The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulg... more The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulgaris cultivated in Tunisian soils. This strain was selected on the basis of its high symbiotic effectiveness in laboratory conditions. In order to assess its ability to compete indigenous rhizobia, this strain was labelled with gusA gene. Conservation of initial effectiveness and competitiveness by transconjugants was tested. A transconjugant was introduced in three soil-core microcosms originating from different geographical and agronomic regions. Nodulation monitoring showed that the labelled transconjugant was able to occupy more than 90% of nodules at 30 days after inoculation. The nodule occupancy by the introduced strain was high even in the soil sample of Mateur which showed an MPN value of 103 rhizobia/g of dry soil. A significant improvement of plant productivity by inoculation was observed with the three soil samples in green house. Field inoculation with the parental strain showed a significant increase in nodule number, pod number and seed dry weight. The improvement of plant productivity in green house or in field conditions was equal or better than nitrogen fertilisation.
A mildly acid-sensitive mutant o f Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced co... more A mildly acid-sensitive mutant o f 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 o f T n 5 and the observed mucoid phenotype, acid sensitivity and TnS-induced kanamycin resistance were 100 O/ O co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid o f a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRl 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 TnS in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti R m l 0 2 l (71-3 O/ O identity over 892 bp). A t the protein level there was 70% identity and 9303% similarity over 267 amino acids with the ExoR protein of R. meliloti RmlO21.
Two ‘calcium-irreparable’ acid-sensitive mutants were identified after mutagenizing Rhizobium leg... more 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 actP gene. 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.
Journal of Molecular Microbiology and Biotechnology, 2004
To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we ha... more 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.
Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse r... more Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.
The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to... more 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.
Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range ... more Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome sequence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a feature unique to this medic microsymbiont. Editorial note -Readers are advised that in Opinion 84 the Judicial Commission of the International Committee on Systematics of Prokaryotes ruled that the genus name Ensifer Casida 1982 has priority over Sinorhizobium Chen et al. 1988 and the names are synonyms [1]. It was further concluded that the transfer of members of the genus Sinorhizobium to the genus Ensifer, as proposed by Young [2] would not cause confusion.
The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulg... more The rhizobia strain R. gallicum bv. gallicum 8a3 was isolated from root nodules of Phaseolus vulgaris cultivated in Tunisian soils. This strain was selected on the basis of its high symbiotic effectiveness in laboratory conditions. In order to assess its ability to compete indigenous rhizobia, this strain was labelled with gusA gene. Conservation of initial effectiveness and competitiveness by transconjugants was tested. A transconjugant was introduced in three soil-core microcosms originating from different geographical and agronomic regions. Nodulation monitoring showed that the labelled transconjugant was able to occupy more than 90% of nodules at 30 days after inoculation. The nodule occupancy by the introduced strain was high even in the soil sample of Mateur which showed an MPN value of 10 3 rhizobia/g of dry soil. A significant improvement of plant productivity by inoculation was observed with the three soil samples in green house. Field inoculation with the parental strain showed a significant increase in nodule number, pod number and seed dry weight. The improvement of plant productivity in green house or in field conditions was equal or better than nitrogen fertilisation.
Rhizob ium le guminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-neg ati... more Rhizob ium le guminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-neg ative, non-spore-forming rod that was isolated from an ineffective root nodule recovered from the roots of the annual clover Trifolium rueppellianum Fresen g rowing in Ethiopia. WSM2012 has a na rrow, specialized host rang e for N 2 -fixation. Here we describe the features of R. leguminosarum bv. trifolii strain WSM2012, tog ether with genome sequence information and annotation. The 7,180,565 bp hig h-quality-draft genome is arrang ed into 6 scaffolds of 68 contig s, contains 7,080 protein-coding g enes and 86 RNA-only encoding genes, and is one of 20 rhizobial g enomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Prog ram.
Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse r... more Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.
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Papers by Ravi Tiwari