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

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 3049-3059 ◽  
Author(s):  
Wayne G. Reeve ◽  
Lambert Bräu ◽  
Joanne Castelli ◽  
Giovanni Garau ◽  
Christian Sohlenkamp ◽  
...  
Keyword(s):  
Cell Growth ◽  
Sinorhizobium Meliloti ◽  
Calcium Concentration ◽  
Transmembrane Protein ◽  
Sequence Similarity ◽  
Membrane Lipid ◽  
Start Codon ◽  
Major Constituent ◽  
Rhizobium Tropici ◽  
Sinorhizobium Medicae

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.

scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

scholarly journals Citrate Synthase Mutants of Sinorhizobium fredii USDA257 Form Ineffective Nodules with Aberrant Ultrastructure

2003 ◽  
Vol 69 (6) ◽  
pp. 3561-3568 ◽  
Author(s):  
Hari B. Krishnan ◽  
Won-Seok Kim ◽  
Jeong Sun-Hyung ◽  
Kil Yong Kim ◽  
Guoqiao Jiang
Keyword(s):  
Sinorhizobium Meliloti ◽  
Citrate Synthase ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Light And Electron Microscopy ◽  
Tca Cycle ◽  
Single Copy ◽  
Membrane Structures ◽  
Rhizobium Tropici ◽  
Sinorhizobium Fredii

ABSTRACT The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.

scholarly journals Cell Growth Inhibition upon Deletion of Four Toxin-Antitoxin Loci from the Megaplasmids of Sinorhizobium meliloti

2013 ◽  
Vol 196 (4) ◽  
pp. 811-824 ◽  
Author(s):  
B. Milunovic ◽  
G. C. diCenzo ◽  
R. A. Morton ◽  
T. M. Finan
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Sinorhizobium Meliloti ◽  
Cell Growth Inhibition

scholarly journals RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (10) ◽  
pp. 4433-4440 ◽  
Author(s):  
N Chiannilkulchai ◽  
R Stalder ◽  
M Riva ◽  
C Carles ◽  
M Werner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Stop Codon ◽  
Sequence Similarity ◽  
Start Codon ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Multicopy Plasmid

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

scholarly journals pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2750-2756 ◽  
Author(s):  
Toshio Yamaguchi ◽  
Fuminori Tsutsumi ◽  
Péter Putnoky ◽  
Masahiro Fukuhara ◽  
Tatsunosuke Nakamura
Keyword(s):  
Sinorhizobium Meliloti ◽  
Sequence Similarity ◽  
Plant Root ◽  
Functional Expression ◽  
Amino Acid Sequence Similarity ◽  
Ph Optimum ◽  
Cation Selectivity ◽  
High Amino Acid ◽  
Alkaline Conditions ◽  
Ph Dependent

The pha1 gene cluster (pha1A′-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K+/H+ antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K+ and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H+ antiporters (Mrp antiporters). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na+, unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H+ antiport activity. We showed that the Pha1 system is indeed a K+/H+ antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na+; this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na+/H+ and K+/H+ antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K+/H+ and Na+/H+ antiport activities of the Pha1 system.

scholarly journals Serine-Threonine Kinases Encoded by SplithipAHomologs Inhibit Tryptophanyl-tRNA Synthetase

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Stine Vang Nielsen ◽  
Kathryn Jane Turnbull ◽  
Mohammad Roghanian ◽  
Rene Bærentsen ◽  
Maja Semanjski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Gene Family ◽  
Sequence Similarity ◽  
Trna Synthetase ◽  
Bacterial Toxin ◽  
Content Type ◽  
Multidrug Tolerance ◽  
K 12

ABSTRACTType II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact.hipBAofEscherichia colistrain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction ofhipAinhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed thattrpSin high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT ofE. coliO127 is a kinase that phosphorylates tryptophanyl-tRNA synthetasein vitroat a conserved serine residue. Consistently, induction ofhipTinhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream fromhipT, calledhipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipSin vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additionalhipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus,hipBSTconstitutes a novel family of tricomponent TA modules wherehipAhas been split into two genes,hipSandhipT, that function as a novel type of TA pair.IMPORTANCEBacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified washipAofEscherichia colistrain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. ThehipAgene encodes the toxin of thehipBATA module, whilehipBencodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family,hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

Pathways for phosphatidylcholine biosynthesis in bacteria

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3461-3471 ◽  
Author(s):  
Fernando Martínez-Morales ◽  
Max Schobert ◽  
Isabel M. López-Lara ◽  
Otto Geiger
Keyword(s):  
Borrelia Burgdorferi ◽  
Legionella Pneumophila ◽  
Bradyrhizobium Japonicum ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Brucella Melitensis ◽  
Membrane Lipid ◽  
Mesorhizobium Loti ◽  
Methylation Pathway ◽  
Phosphatidylcholine Biosynthesis

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

scholarly journals Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases.

1990 ◽  
Vol 110 (3) ◽  
pp. 669-679 ◽  
Author(s):  
L Bomblies ◽  
E Biegelmann ◽  
V Döring ◽  
G Gerisch ◽  
H Krafft-Czepa ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Fusion Gene ◽  
Sequence Similarity ◽  
Transcript Level ◽  
Crystal Protein ◽  
Major Constituent ◽  
Crystalline Inclusion ◽  
Developmentally Regulated ◽  
Sequence Similarities ◽  
D2 Protein

Developing cells of Dictyostelium discoideum contain crystalline inclusion bodies. The interlattice spaces of the crystals are approximately 11 nm, and their edge dimensions vary in aggregating cells from 0.1 to 0.5 micron. The crystals are enclosed by a membrane with the characteristics of RER. To unravel the nature of the crystals we isolated them under electron microscopical control and purified the two major proteins that cofractionate with the crystals, one of an apparent molecular mass of 69 kD, the other of 56 kD. This latter protein proved to be identical with the protein encoded by the developmentally regulated D2 gene of D. discoideum, as shown by its reactivity with antibodies raised against the bacterially expressed product of a D2 fusion gene. The D2 gene is known to be strictly regulated at the transcript level and to be controlled by cAMP signals. Accordingly, very little of the 56-kD protein was detected in growth phase cells, maximal expression was observed at the aggregation stage, and the expression was stimulated by cAMP pulses. The 69-kD protein is the major constituent of the crystals and is therefore called "crystal protein." This protein is developmentally regulated and accumulates in aggregating cells similar to the D2 protein, but is not, or is only slightly regulated by cAMP pulses. mAbs specific for either the crystal protein or the D2 protein, labeled the intracellular crystals as demonstrated by the use of immunoelectron microscopy. The complete cDNA-derived amino acid sequence of the crystal protein indicates a hydrophobic leader and shows a high degree of sequence similarity with Torpedo acetylcholinesterase and rat lysophospholipase. Because the D2 protein also shows sequence similarities with various esterases, the vesicles filled with crystals of these proteins are named esterosomes.

scholarly journals Identification of Genes in the RosR Regulon ofRhizobium etli

2000 ◽  
Vol 182 (6) ◽  
pp. 1706-1713 ◽  
Author(s):  
Mark A. Bittinger ◽  
Jo Handelsman
Keyword(s):  
Cell Surface ◽  
Sinorhizobium Meliloti ◽  
Sequence Similarity ◽  
Surface Characteristics ◽  
Nucleotide Sequence Analysis ◽  
Plant Host ◽  
Glucuronidase Activity ◽  
Nodulation Competitiveness ◽  
Mutant Derivative ◽  
Mass Mating

ABSTRACT RosR is a determinant of nodulation competitiveness and cell surface characteristics of Rhizobium etli and has sequence similarity to a family of transcriptional repressors. To understand how RosR affects these phenotypes, we mutagenized a rosR mutant derivative of R. etli strain CE3 with a mini-Tn5 that contains a promoterless gusA gene at one end, which acts as a transcriptional reporter. Using a mass-mating technique, we introduced rosR into each mutant in trans and screened for mutants that expressed different levels of β-glucuronidase activity in the presence and absence ofrosR. A screen of 18,000 mutants identified 52 insertions in genes negatively regulated by RosR and 1 insertion in a gene positively regulated by RosR. Nucleotide sequence analysis of the regions flanking the insertions suggests that RosR regulates genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism and those in a region containing sequence similarity to virC1 and virD3 fromAgrobacterium tumefaciens. Two of the mutants produced colonies with altered morphology and were more competitive in nodulation than was CE3ΔrosR, the rosRparent. One mutant that contained an insertion in a gene with similarity to exsH of Sinorhizobium melilotidid not nodulate the plant host Phaseolus vulgaris withoutrosR. These results indicate that RosR directly or indirectly influences expression of diverse genes in R. etli, some of which affect the cell surface and nodulation competitiveness.

scholarly journals A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

1992 ◽  
Vol 12 (3) ◽  
pp. 1162-1178 ◽  
Author(s):  
C Costigan ◽  
S Gehrung ◽  
M Snyder
Keyword(s):  
Cell Growth ◽  
Vegetative Growth ◽  
Sequence Similarity ◽  
Growth Control ◽  
Cell Morphogenesis ◽  
Wild Type ◽  
Extra Copy ◽  
Synthetic Lethal ◽  
Cell Biol ◽  
Regulatory Link

The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes.

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