Exo Mutants Research Articles

Biochemical characterization of glucoamylase from the hyperproducerexo-1 mutant strain ofNeurospora erassa

The Neurospora crassa exo-1 mutant produced maximum extracellular glucoamylase activity in media supplemented with starch as the sole carbon source. The apparent molecular mass of the enzyme was 82 kDa (SDS-PAGE and gel filtration). The enzyme was a glycoprotein with 5.1 % carbohydrate content and exhibited a temperature optimum of 60 °C. The pH optima were 5.4 and 5.0 for glucoamylase and maltase activities, respectively. Cu2+ inhibited maltase activity while Mn2+ stimulated glucoamylase activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch was the best substrate utilized and amylose was hydrolyzed faster than maltose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.

Defective Infection and Nodulation of Clovers by Exopolysaccharide Mutants of Rhizobium leguminosarum bv. trifolii

The biological and symbiotic properties of two types of exopolysaccharide deficient (Exo- phenotype) mutants of Rhizobium leguminosarum were investigated. One mutant, a pss1 derivative (ANU437) of R. l. bv. trifolii, produces very low levels of acidic exopolysaccharide (EPS) and of the acidic oligosaccharide (AOS) repeating unit lacking its O-acetyl substituent. The second strain, originally a R. I. bv. viciae derivative, but containing a clover pSym plasmid (Exo-1 mutant ANU54(pBRIAN)), did not produce any EPS polymer or detectable oligosaccharide repeating unit. The Exo- mutants formed no capsules and their growth in laboratory media was comparable to their parent strains but was inhibited by the addition of the phytoalexins medicarpin and kievitone, but not by the phytoalexin pisatin. Both Exo- mutants were slow to induce clover root hair curling (Hac+), and formed growing infection threads (Inf-) only on white clovers. However, the extent of these early symbiotic defects depended on the plant growth conditions used. Both mutants induced small non-nitrogen-fixing (Fix-) nodules on white and subterranean clovers. Within the white clover nodules induced by mutant ANU437, bacteria were released into plant cells. On white clovers at elevated light and temperature conditions, the Exo-1 mutant ANU54(pBRIAN) usually failed to initiate infection threads and thus formed empty nodules which do not contain any bacteria. In addition, this mutant formed only empty nodules without infection threads on subterranean clovers under all growth conditions tested. The outermost layers of plant cells in these empty nodules had thickened cell walls and deposited materials. These experiments show that EPS synthesis is essential for the induction of rapid root hair curling, proper infection thread formation and the ability to sustain the growth of induced nodules on various clovers.

A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.

Exonuclease I (Exo I) from Schizosaccharomyces pombe, a 5'-->3' double-stranded DNA exonuclease, is induced during meiotic prophase I. The exo1 gene is a member of a family of related DNA repair genes, including RAD2/rad13/xpgc and YKL510/rad2, conserved from yeast to humans. An exo1 mutant displays a mutator phenotype and alters activity of the ade6-M387 marker effect. These results suggest that Exo I acts in a pathway that corrects mismatched base pairs.

Pleiotropic effects of regulatory ros mutants of Agrobacterium radiobacter and their interaction with Fe and glucose.

Four exo mutants of Agrobacterium radiobacter, defective in the synthesis of acidic exopolysaccharide were complemented by a gene from that species, which is similar to the transcriptional regulator, ros, of A. tumefaciens. It was confirmed that this A. radiobacter gene, which we term rosAR, like ros, repressed its own transcription as well as that of virC and virD, two loci involved in tumorigenesis. The sequence of RosAR suggested that it might bind to a transition metal and its repressor abilities were shown to require Fe in the medium; repression was also enhanced with increasing levels of glucose. Certain rosAR mutants, in which its 3' end was removed were dominant; i.e., when plasmids containing such mutant forms of the gene were introduced into wild-type A. radiobacter, the transconjugants were nonmucoid. Such effects were also seen in a wide range of bacteria, including Escherichia coli and Xanthomonas. Several mutants that were complementd by rosAR also accumulated protoporphyrin, suggesting a defect in haem synthesis.

Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

The exo genes of Rhizobium meliloti are needed for the synthesis of an acidic exopolysaccharide, succinoglycan. We have assigned biosynthetic roles to the products of the exo genes by characterizing succinoglycan biosynthetic intermediates from exo mutant strains. We propose a model of succinoglycan biosynthesis in which the products of the exoY and exoF genes function in the addition of the first sugar, galactose, to the lipid carrier; the products of the exoA, exoL, exoM, exoO, exoU, and exoW genes function in subsequent sugar additions; and the product of the exoV gene functions in the addition of pyruvate. The products of the exoP, exoQ, and exoT genes are required for polymerization of the octasaccharide subunits or transport of the completed polymer.

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.

Acidic conditions permit effective nodulation of alfalfa by invasion-deficient Rhizobium meliloti exoD mutants.

Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.

Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.

Identification of the exo loci required for exopolysaccharide synthesis in Agrobacterium radiobacter NCIB 11883

We initiated a genetic analysis of Agrobacterium radiobacter NCIB 11883 with particular reference to the (exo) genes required for exopolysaccharide synthesis. Following mutagenesis with nitrosoguanidine, several exo mutant strains were isolated and several of the mutations were corrected by DNA cloned in a newly constructed cosmid library. Analysis of various complementing cosmids by genetic and physical criteria indicated that exo loci were quite widely dispersed in the bacterial genome. Certain exo mutations were corrected by different cosmids that shared no homologous DNA; possible explanations for this are presented. Using phoA fusions, it was shown that some exo genes were, or were closely linked to, genes that specified polypeptides associated with the bacterial cell surface. By introducing the cloned exo genes of Rhizobium meliloti it was found that only one out of thirty exo mutants of A. radiobacter was corrected by a defined exo locus of the former species; further analysis indicated that this particular exo gene corresponded to exoB of R. meliloti. Finally, it was found that several A. radiobacter exo mutants were non-mucoid on media with dicarboxylic acids as sole carbon source but appeared to be wild-type when sugars were the source of carbon.

LpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.

Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide

Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen. This Fix- phenotype can be suppressed by an R. meliloti Rm41 gene that affects lipopolysaccharide structure. Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains. Two other lps mutations isolated previously from SU47 also prevented suppression.

Nodulin Gene Expression and ENOD2 Localization in Effective, Nitrogen-Fixing and Ineffective, Bacteria-Free Nodules of Alfalfa.

Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R. meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( 1-naphthyl)phthalamic acid or 2,3,5-triiodobenzoic acid. These bacteria-free nodules contain transcripts for the nodulins Nms30 and MsENOD2; transcripts for late nodulins like leghemoglobin are not detected. In situ hybridization studies demonstrated that ENOD2 transcripts were localized in parenchyma cells at the base and along the periphery of nitrogen-fixing alfalfa root nodules. The ENOD2 gene was also expressed in a tissue-specific manner in nodules elicited by N-( 1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid. In bacteria-free nodules induced by R. meliloti exo mutants and A. tumefaciens transconjugants carrying either one or both R. meliloti symbiotic plasmids, ENOD2 transcripts were also detected but were usually localized to parenchyma cells at the base instead of along the periphery of the nodule. On the basis of the pattern of ENOD2 gene expression, we conclude that the developmental pathway of bacteria-free nodules, whether bacterially or chemically induced, is the same as that of nitrogen-fixing nodules, and, furthermore, that the auxin transport inhibitors in their action mimic some factor(s) that trigger nodule development.

Two genes that regulate exopolysaccharide production in Rhizobium meliloti

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.

Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.