ORCID Profile
0000-0003-3354-770X
Current Organisation
Murdoch University
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Publisher: Microbiology Society
Date: 06-1997
DOI: 10.1099/00221287-143-6-1951
Abstract: 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.
Publisher: Springer Netherlands
Date: 1998
Publisher: Springer Netherlands
Date: 2000
Publisher: Microbiology Society
Date: 10-2006
Abstract: 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.
Publisher: Wiley
Date: 04-1997
Abstract: Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif. The molecular characterization of these proteins has been h ered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (delta epsilon 248, 24 mM-1 cm-1) indicated the presence of a Cd(Cys-S)4 center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD, app 3-4 microM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between-70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase alpha-subunit. A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.
Publisher: American Society for Microbiology
Date: 16-03-2023
DOI: 10.1128/MRA.01215-22
Abstract: Pseudomonas syringae MUP17 was isolated from Western Australian frost-damaged barley. The MUP17 complete genome contained a 5,850,185-bp single circular chromosome with a GC content of 59.12%. IMG/M genome annotation identified 5,012 protein-coding genes, 1 of which encoded an ice-nucleation protein containing 19 occurrences of a highly repetitive PF00818 domain.
Publisher: Wiley
Date: 28-09-2007
Publisher: Microbiology Society
Date: 07-1996
DOI: 10.1099/13500872-142-7-1693
Abstract: An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (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 lacZ expression vector and measuring beta-galactosidase 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.
Publisher: Microbiology Society
Date: 12-1998
DOI: 10.1099/00221287-144-12-3335
Abstract: Summary: 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 Zn 2+ , Cu 2+ , H 2 O 2 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 lify a 3�5 kb Bam HI fragment containing phrR from the mutant S. meliloti TG2-6 ( actA ::Tn5). DNA fragments from a Bam HI/ Sal I digest of the lified 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.
Publisher: Wiley
Date: 09-01-2017
DOI: 10.1111/MMI.13592
Abstract: 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.
Publisher: American Society for Microbiology
Date: 16-03-2023
DOI: 10.1128/MRA.01275-22
Abstract: Pseudomonas syringae MUP20 was isolated from Western Australian frost-damaged wheat. The MUP20 complete genome contained a 6,045,198-bp single circular chromosome with a GC content of 59.03%. IMG/M genome annotation identified 5,245 protein-coding genes, 1 of which encoded an ice nucleation protein containing 20 occurrences of a highly repetitive PF00818 domain.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Elsevier
Date: 2010
Publisher: Springer Netherlands
Date: 2002
Publisher: Wiley
Date: 02-2002
DOI: 10.1046/J.1365-2958.2002.02791.X
Abstract: 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.
Publisher: Microbiology Society
Date: 15-07-2009
Abstract: Biserrula pelecinus L. is a pasture legume that was introduced to Australia from the Mediterranean basin in 1993. Although the native rhizobial population could not nodulate B. pelecinus at the time of its introduction, recent research has shown the emergence of a ersity of strains (novel isolates) that are able to do so. Three novel isolates, WSM2073T, WSM2074 and WSM2076, had nearly identical 16S rRNA gene sequences, and clustered separately with all recognized species of the genus Mesorhizobium. Conversely, the novel isolate WSM2075T had >23 nt mismatches with the above three isolates. All four novel isolates shared 97-99% 16S rRNA gene sequence similarity with the type strains of all recognized Mesorhizobium species. However, strains WSM2073T, WSM2074 and WSM2076 showed <95.2% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species, and <92.9% to WSM2075T (which also shared <95.5% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species). Results for GSII gene sequencing were consistent with those for the dnaK gene. The fatty acid profiles of the novel isolates were diagnostic of root-nodule bacteria, but did not match those of recognized bacterial species. Strain WSM2075T had a significantly different fatty acid profile from the other three isolates. The above results indicated that strains WSM2073T, WSM2074 and WSM2076 represent the same species. Strain WSM2073T showed <45% DNA-DNA relatedness and WSM2075T<5% DNA-DNA relatedness with the type strains of recognized Mesorhizobium species these two novel isolates shared 59% DNA-DNA relatedness. Collectively, these data indicate that strains WSM2073T, WSM2074 and WSM2076, and strain WSM2075T belong to two novel species of the genus Mesorhizobium, for which the names Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov. are proposed, respectively. The type strain of Mesorhizobium australicum sp. nov. is WSM2073T (=LMG 24608T=HAMBI 3006T) and the type strain of Mesorhizobium opportunistum sp. nov. is WSM2075T (=LMG 24607T=HAMBI 3007T).
Publisher: Springer Science and Business Media LLC
Date: 18-01-2009
DOI: 10.1007/S00203-009-0456-0
Abstract: 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 lification 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.
Publisher: Microbiology Society
Date: 05-2007
Abstract: Biserrula pelecinus L. is a pasture legume species that forms a highly specific nitrogen-fixing symbiotic interaction with a group of bacteria that belong to Mesorhizobium . These mesorhizobia have .8 % sequence similarity to Mesorhizobium ciceri and Mesorhizobium loti for the 16S rRNA gene (1440 bp) and .3 % sequence similarity to M. ciceri for the dnaK gene (300 bp), and strain WSM1271 has 100 % sequence similarity to M. ciceri for GSII (600 bp). Strain WSM1271 had 85 % relatedness to M. ciceri LMG 14989 T and 50 % relatedness to M. loti LMG 6125 T when DNA–DNA hybridization was performed. WSM1271 also had a similar cellular fatty acid profile to M. ciceri . These results are strong evidence that the Biserrula mesorhizobia and M. ciceri belong to the same group of bacteria. Significant differences were revealed between the Biserrula mesorhizobia and M. ciceri in growth conditions, antibiotic resistance and carbon source utilization. The G+C content of the DNA of WSM1271 was 62.7 mol%, compared to 63–64 mol% for M. ciceri . The Biserrula mesorhizobia contained a plasmid (~500 bp), but the symbiotic genes were detected on a mobile symbiosis island and considerable variation was present in the symbiotic genes of Biserrula mesorhizobia and M. ciceri . There was .6 % sequence similarity for nodA and .9 % for nifH between Biserrula mesorhizobia and M. ciceri . Moreover, the Biserrula mesorhizobia did not nodulate the legume host of M. ciceri , Cicer arietinum , and M. ciceri did not nodulate B. pelecinus . These significant differences observed between Biserrula mesorhizobia and M. ciceri warrant the proposal of a novel biovar for Biserrula mesorhizobia within M. ciceri . The name Mesorhizobium ciceri biovar biserrulae is proposed, with strain WSM1271 (=LMG 23838=HAMBI 2942) as the reference strain.
Publisher: CSIRO Publishing
Date: 2001
DOI: 10.1071/EA99155
Abstract: Bacteria face a variety of problems in trying to survive and grow in acidic environments. These include maintaining intracellular pH (pHi) in order to protect internal cell components, modifying or abandoning those external structures inevitably exposed to acidity, and resisting stresses whose interaction with pH may be the actual determinant of survival or growth rather than H+ toxicity per se. An important aspect of acid resistance in Gram-negative bacteria (including the root nodule bacteria) is the adaptive acid tolerance response (ATR), whereby cells grown at moderately acid pH are much more resistant to being killed under strongly acidic conditions than are cells grown at neutral pH. Survival during pH shock is also markedly affected by the calcium concentration in the medium. The pH at which commercial legume inoculants are grown and supplied for inoculation into acid soils may therefore be of considerable importance for initial inoculant survival. The mechanisms of resistance to acidity in root nodule bacteria have been investigated via 3 approaches: (i) creation of acid-sensitive mutants from acid-tolerant strains, and identification of the genes involved (ii) random insertion of reporter genes to create mutants with pH-dependent reporter expression and (iii) proteomics and identification of proteins regulated in response to acidity. The results of the first approach, directed at genes essential for growth at acid pH, have identified a sensor–regulator gene pair (actS–actR), a copper-transporting ATPase (actP), and another gene involved in lipid metabolism (actA), inactivation of which results in sensitivity to heavy metals. While the ActS–ActR system is undoubtedly required for both acid tolerance and the ATR, it is also involved in global regulation of a wide range of cellular processes. The second approach has allowed identification of a range of acid-responsive genes, which are not themselves critical to growth at low pH. One of these (phrR) is itself a regulator gene induced by a range of stresses including acid pH, but not controlled by the ActS–ActR system. Another, lpiA, responds specifically to acidity (not to other stresses) and may well be an antiporter related to nhaB, which is involved in Na+ transport in other bacteria. The third approach indicates a number of proteins whose concentration changes with a switch from neutral to acidic growth pH most of these seem to have no homologues in the protein databases, while the blocked N-terminal sequences of others have prevented identification. It has been common experience that strains of root nodule bacteria selected for acid tolerance in the laboratory are not necessarily successful as inoculants in acid soils. In the light of the complex interactive effects on growth and survival of H+, Ca2+ and Cu2+ concentrations in our studies, this lack of correlation is no longer surprising. It remains to be seen whether it will be possible to improve the correlation between growth on laboratory media and performance in acid soils by determining which strains show an ATR, and by screening on media with defined ranges of concentration of some of these critical metal ions, perhaps approximating those to be expected in the soils in question.
Publisher: Microbiology Society
Date: 03-1996
DOI: 10.1099/13500872-142-3-601
Abstract: The actA gene, which is disrupted by Tn 5 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 pI 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.
Publisher: Wiley
Date: 14-04-2008
DOI: 10.1111/J.1469-8137.2008.02464.X
Abstract: DOI: 10.1111/j.1469-8137.2008.02494.x
Publisher: Wiley
Date: 27-06-2007
DOI: 10.1111/J.1462-2920.2007.01368.X
Abstract: 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 N(2) fixation. This study investigated the development of rhizobial ersity 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 N(2) 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 erse strains dominated nodule occupancy indicating recently evolved strains are highly competitive. All isolates tested were less effective and six were ineffective in N(2) fixation. Twelve randomly selected erse 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 N(2) fixation on B. pelecinus following the lateral transfer of a symbiosis island from inoculants to other soil bacteria.
Publisher: Oxford University Press (OUP)
Date: 28-03-2017
DOI: 10.1093/NAR/GKX209
Publisher: Springer Science and Business Media LLC
Date: 10-2005
Publisher: Wiley
Date: 16-02-2021
DOI: 10.1111/GFS.12518
Publisher: Springer Science and Business Media LLC
Date: 25-02-2012
Publisher: Microbiology Society
Date: 11-2001
DOI: 10.1099/00207713-51-6-1983
Abstract: Three bacterial strains (WSM 1283, WSM 1284, WSM 1497) isolated from root nodules of the pasture legume Biserrula pelecinus L. growing in Morocco, Italy and Greece, respectively, were studied in order to determine their phylogenetic relationship to the other members of the family Rhizobiaceae. A polyphasic approach, which included analyses of morphological and physiological characteristics, plasmid profiles, symbiotic performance and 16S rRNA gene sequencing, indicated that these strains belong to the genus Mesorhizobium.
Publisher: Elsevier BV
Date: 05-1993
Publisher: American Society for Microbiology
Date: 16-03-2023
DOI: 10.1128/MRA.01276-22
Abstract: The genome of Pseudomonas syringae MUP32, which was isolated from frost-damaged pea in New South Wales, Australia, is tripartite and contains a circular chromosome (6,032,644 bp) and two plasmids (61,675 and 54,993 bp). IMG/M genome annotation identified 5,370 protein-coding genes, one of which encoded an ice-nucleation protein with 19 repetitive PF00818 domains.
Publisher: Springer Netherlands
Date: 2008
Publisher: S. Karger AG
Date: 2004
DOI: 10.1159/000078657
Abstract: To elucidate the mechanisms of pH response in an acid-tolerant i Sinorhizobium medicae /i 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 i S. medicae /i 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 i S. medicae /i .
Publisher: S. Karger AG
Date: 2004
DOI: 10.1159/000078656
Abstract: The low pH sensitivity of i Sinorhizobium /i species is one of the major causes of reduced productivity of i Medicago /i species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant i Sinorhizobium medicae /i strain, a pool of random promoter fusions to i gusA /i 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 i Sinorhizobium meliloti /i strain 1021. Acid activated expression was detected for i fixNO /i , i kdpC /i , i lpiA /i , and i hrR /i 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 i S. medicae /i .
Publisher: Microbiology Society
Date: 06-1999
DOI: 10.1099/13500872-145-6-1307
Abstract: 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.
Publisher: American Society for Microbiology
Date: 11-2006
DOI: 10.1128/AEM.00889-06
Abstract: 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.
Publisher: Springer Netherlands
Date: 2008
No related grants have been discovered for Ravi Tiwari.