Expression of Rhizobium leguminosarum CFN42 genes for lipopolysaccharide in strains derived from different R. leguminosarum soil isolates.

Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated. The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides. Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants. One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition. The other mutant was complemented by a second CFN42 lps genetic region. In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells. Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R. leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure. Transfer of these two genetic regions into wild-type strains derived from R. leguminosarum ANU843 and 128C53 gave results consistent with this conclusion. The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843.     

Varying the abundance of O antigen in Rhizobium etli and its effect on symbiosis with Phaseolus vulgaris

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region alpha, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region alpha elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region alpha and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.     

Studies on the pathophysiological mechanism of Campylobacter jejuni-induced fluid secretion in rat ileum

Abstract Lipopolysaccharide (LPS) patterns were obtained from fast-growing Rhizobium strains after silver staining of proteinase K treated cells lysates, run in SDS-PAGE. The rhizobia came from root nodules of Acacia senegal and Prosopis chilensis , collected in differents part of the Sudan. The LPS profiles of all strains were typical of rhizobia. Two different LPS region with lower and higher electrophoretic mobility (region I and region II, respectively) coulld be dinguished in the gels, and based on the profiles the strains were divided into three groups. Strains isolated from A. senegal showed a wider range of different profiles than strains isolated from P. chilensis , even though the plants belong to the same cross-infection group. Otherwise there was no clear correlation between the taxonomic relatedness or site of isolation of the strains and their LPS profiles.     

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.     

Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts

Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture. The mutants were classified into three groups. Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome. The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment. Attempts to complement mutants of this second group with cloned DNA were unsuccessful. The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants. The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants. The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants. Mutants in each of these groups were mated with R. leguminosarum strains that nodulated peas (R. leguminosarum biovar viciae) or clovers (R. leguminosarum biovar trifolii). Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R. leguminosarum biovar viciae or R. leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases. These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.     

Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier

Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

Summary A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.     

Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa.

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.     

Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.     

Transfectability of rough strains of Salmonella typhimurium.

Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection.     

L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product

Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments. Therefore, the genes encoding asparaginases I and II are highly sequence related.     

A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding

The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDDelta107C (deletion of the last 107 C-terminal amino acids), UvrDDelta102C, and UvrDDelta40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDDelta107C and UvrDDelta102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDDelta40C protein fully complemented the loss of helicase II in both repair pathways. UvrDDelta102C and UvrDDelta40C were purified to apparent homogeneity and characterized biochemically. UvrDDelta102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (kcat = 0.01% of the wild-type level). UvrDDelta40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Decreased symbiotic effectiveness of Rhizobium leguminosarum strains carrying plasmid RP4

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncPα classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.