ThephbC(poly-β-hydroxybutyrate synthase) gene ofRhizobium(Sinorhizobium)melilotiand characterization ofphbCmutants

1998 ◽  
Vol 44 (6) ◽  
pp. 554-564 ◽  
Author(s):  
Laura B Willis ◽  
Graham C Walker
Keyword(s):  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Alcaligenes Eutrophus ◽  
Wild Type ◽  
Polypeptide Sequence ◽  
Phb Production ◽  
Phb Synthase ◽  
Insertion Mutations ◽  
Phbc Gene

Defined insertion mutations have been constructed in theRhizobium (Sinorhizobium) meliloti phbC gene, which encodes poly-β-hydroxybutyrate (PHB) synthase. The locus was isolated and subcloned from a genomic library of R. meliloti Rm1021 by complementation of a phbC mutation of Alcaligenes eutrophus. PHB production was detected in wild-type R. meliloti under nutrient-limited conditions but not in rich medium. No PHB production was detected in the R. meliloti phbC mutants. The DNA sequence of the R. meliloti phbC gene was determined. The deduced polypeptide sequence is homologous to previously identified PhbCs from other bacteria. The R. meliloti phbC locus maps to pRmeSU47a, the smaller of the two megaplasmids in this strain.Key words: Rhizobium meliloti, PHB, PHA, poly-β-hydroxybutyrate, phbC.

scholarly journals The Succinyl and Acetyl Modifications of Succinoglycan Influence Susceptibility of Succinoglycan to Cleavage by the Rhizobium meliloti Glycanases ExoK and ExsH

1998 ◽  
Vol 180 (16) ◽  
pp. 4184-4191 ◽  
Author(s):  
Gregory M. York ◽  
Graham C. Walker
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Meliloti ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Mutant Strains ◽  
Acetyl Group

ABSTRACT In Rhizobium meliloti (Sinorhizobium meliloti) cultures, the endo-1,3-1,4-β-glycanases ExoK and ExsH depolymerize nascent high-molecular-weight (HMW) succinoglycan to yield low-molecular-weight (LMW) succinoglycan. We report here that the succinyl and acetyl modifications of succinoglycan influence the susceptibility of succinoglycan to cleavage by these glycanases. It was previously shown that exoH mutants, which are blocked in the succinylation of succinoglycan, exhibit a defect in the production of LMW succinoglycan. We have determined that exoZ mutants, which are blocked in the acetylation of succinoglycan, exhibit an increase in production of LMW succinoglycan. For both wild-type andexoZ mutant strains, production of LMW succinoglycan is dependent on the exoK + andexsH + genes, implying that the ExoK and ExsH glycanases cleave HMW succinoglycan to yield LMW succinoglycan. By supplementing cultures of glycanase-deficient strains with exogenously added ExoK or ExsH, we have demonstrated directly that the absence of the acetyl group increases the susceptibility of succinoglycan to cleavage by ExoK and ExsH, that the absence of the succinyl group decreases the susceptibility of succinoglycan to cleavage, and that the succinyl effect outweighs the acetyl effect for succinoglycan lacking both modifications. Strikingly, nonsuccinylated succinoglycan actually can be cleaved by ExoK and ExsH to yield LMW succinoglycan, but only when the glycanases are added to cultures at greater than physiologically relevant concentrations. Thus, we conclude that the molecular weight distribution of succinoglycan in R. meliloti cultures is determined by both the levels of ExoK and ExsH glycanase expression and the susceptibility of succinoglycan to cleavage.

scholarly journals Phosphate Assimilation in Rhizobium(Sinorhizobium) meliloti: Identification of apit-Like Gene

1998 ◽  
Vol 180 (16) ◽  
pp. 4219-4226 ◽  
Author(s):  
Sylvie D. Bardin ◽  
Ralf T. Voegele ◽  
Turlough M. Finan
Keyword(s):  
Transport System ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Meliloti ◽  
Root Nodules ◽  
Phosphate Transport ◽  
Wild Type ◽  
Transcription Start ◽  
Present Evidence ◽  
Suppressor Mutations ◽  
Mutant Cells

ABSTRACT Rhizobium meliloti mutants defective in thephoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix−). We have previously reported that two classes of second-site mutations can suppress the Fix− phenotype ofphoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that theorfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pibut repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pitexpression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increasesorfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDETmutants.

scholarly journals Cloning, Sequencing, and Characterization of thecgmB Gene of Sinorhizobium meliloti Involved in Cyclic β-Glucan Biosynthesis

1999 ◽  
Vol 181 (15) ◽  
pp. 4576-4583 ◽  
Author(s):  
Ping Wang ◽  
Cheryl Ingram-Smith ◽  
Jill A. Hadley ◽  
Karen J. Miller
Keyword(s):  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Functional Expression ◽  
Open Reading Frames ◽  
Inverse Pcr ◽  
Insertion Mutant ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known asRhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transformRhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated thecgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

Regulation of Phosphate Assimilation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1689-1700 ◽  
Author(s):  
Sylvie D Bardin ◽  
Turlough M Finan
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Phosphate Transport ◽  
Wild Type ◽  
Pi Transport ◽  
Regulatory Systems ◽  
Mutant Strains ◽  
Insertion Mutations

Abstract We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF) encoded phosphate transport system grow slowly in media containing 2 mm Pi, and form nodules which fail to fix nitrogen (Fix−). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mm Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.

scholarly journals DctBD-Dependent and -Independent Expression of the Sinorhizobium (Rhizobium) meliloti C4-Dicarboxylate Transport Gene (dctA) During Symbiosis

1998 ◽  
Vol 11 (9) ◽  
pp. 878-886 ◽  
Author(s):  
Bert Boesten ◽  
Jacques Batut ◽  
Pierre Boistard
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Meliloti ◽  
Dicarboxylic Acids ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Coding Region ◽  
Dicarboxylate Transport

The Sinorhizobium meliloti C4-dicarboxylate transport gene (dctA) is essential for symbiotic nitrogen fixation. Under free-living conditions, the expression of dctA is fully dependent on the cognate regulatory genes dctBD. However, during symbiosis with the Medicago sativa host plant, the dctA gene is efficiently expressed even in the absence of the dctBD genes. The spatial expression of the dctA gene has been monitored in situ in mature nitrogen-fixing nodules formed by wild-type and dctD mutant strains. In nodules induced by a wild-type strain, expression was observed in both the infection zone and the nitrogen-fixing zone of the nodule. DctD-independent expression of dctA was observed with a previously described dctA∷lacZ fusion (pCU700) and was found to be confined to the fixation zone (zone III) of mature nodules. Therefore, the operation of the alternative system of symbiotic dctA activation (ASA) is concomitant with the onset of nitrogen fixation, which could be consistent with an increased need for transport of (C)4(-dicarboxylic acids by the nitrogen-fixing) bacteroids. Sequences in the 5′ part of the dctA coding region were found to be essential for the activation of the dctA∷lacZ gene fusions by the ASA. Deletion of these sequences resulted in gene fusions that were found to be strictly dependent on dctBD for expression, under all conditions tested including symbiosis. Such gene fusions allowed us to establish that the DctBD-dependent dctA expression was occurring throughout the whole nodule.

Homology between the genes of octopine catabolism of Rhizobium meliloti A3 and corresponding genes from the Ti plasmid

1993 ◽  
Vol 39 (11) ◽  
pp. 1041-1050 ◽  
Author(s):  
Janique Bergeron ◽  
Carole Beaulieu ◽  
Roger C. Levesque ◽  
Adam Kondorosi ◽  
Patrice Dion
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Ti Plasmid ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Plasmid Genes ◽  
Ornithine Cyclodeaminase

The ability to catabolize crown-gall opines is found in Agrobacterium tumefaciens and various types of nonagrobacteria. Among the 75 Rhizobium meliloti strains tested in this work, 6 utilized the opines octopine and octopinic acid as the sole carbon and nitrogen source. From a genomic library of one of these six strains, R. meliloti A3, a clone conferring the octopine catabolism (Occ) phenotype was identified and named pJMA. A different Occ clone, which had been obtained from R. meliloti Rm41, did not hybridize with clone pJMA from strain A3. However, some fragments of clone pJMA hybridized to a 20-kilobase KpnI fragment containing the Ti plasmid genes of octopine catabolism (occ genes) from A. tumefaciens 15955. Shorter probes carrying the octopine permease genes or part of the octopine oxidase and ornithine cyclodeaminase genes from A. tumefaciens also hybridized with pJMA. The R. meliloti DNA carried by pJMA was localized to a megaplasmid of the wild-type strain A3. Thus, it appears possible that genes represented on the Occ clone from strain A3 share a common origin with the corresponding genes from the Ti plasmid.Key words: Rhizobium meliloti, Agrobacterium tumefaciens, octopine catabolism.

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 The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals Improved fermentation strategies in a bioreactor for enhancing poly(3-hydroxybutyrate) (PHB) production by wild type Cupriavidus necator from fructose

Heliyon ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. e05979
Author(s):  
Daiana Nygaard ◽  
Oxana Yashchuk ◽  
Diego G. Noseda ◽  
Beatriz Araoz ◽  
Élida B. Hermida
Keyword(s):  
Cupriavidus Necator ◽  
Wild Type ◽  
Phb Production

Expression of the nodulation and nitrogen fixation genes in Rhizobium meliloti during development

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 354-360 ◽  
Author(s):  
San Chiun Shen ◽  
Shui Ping Wang ◽  
Guan Qiao Yu ◽  
Jia Bi Zhu
Keyword(s):  
Nitrogen Fixation ◽  
Rhizobium Meliloti ◽  
Abnormal Development ◽  
Wild Type ◽  
Free Living ◽  
Nod Genes ◽  
Nodule Initiation ◽  
Fix Genes ◽  
Nitrogen Fixation Genes

Genes that specify nodulation (nod genes) are only active in the free-living rhizobia or in the nodule initiation state of rhizobia. As soon as the repression of nod genes occurs in the bacteroids of the nodule, nifA is induced, while ntrC is inactivated and thus the nifA-mediated nif/fix genes are turned on. Limitation of available oxygen brings about the induction of nifA, which reflects the actual status of nif/fix gene activities in symbiotic state of rhizobia. Oxygen thus appears to be a major symbiotic signal to the expression of bacteroid nif/fix genes. Mutation of nifA or shortage of nifA product in wild-type rhizobia caused by the inhibition of multicopy nifH/fixA promoters leads to an abnormal development of nodules and premature degradation of bacteroids in nodules.Key words: nitrogen fixation, nodulation, nif/fix regulation, nifA mutant.

Sign in / Sign up

Export Citation Format

Share Document