Thesis (M. Sc.)(Microbiology)--University of Pretoria, 2005. / Includes bibliographical references. Available on the Internet via the World Wide Web.

Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility. Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle. A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation. The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.

The primary objective of this study was to investigate the use of water table management (WTM) as a microbial delivery system for in-situ bioaugmentation of contaminated soils. In addition, the use of Rhizobium ( R.) for PCB degradation in soils was evaluated. / First, the presence and isolation of a variety of strains of Rhizobium meliloti was demonstrated using plant nodulation tests on alfalfa plants in soils that were contaminated for over 15 years with PCBs, PAHs and heavy metals. Next, R. meliloti, strain A-025, was selected based on its membrane (hydrophobicity, adhesion) characteristics and its potential to transform PCBs. This strain was delivered and implanted in sod columns, 200 mm in diameter x 1000 mm in length, packed with a sandy loam soil, using surface and subirrigation. The results of this study showed that subirrigation led to a higher number and a more uniform distribution of the bacterial cells in the soil at 60, 300, 500, and 700 mm depths, than surface irrigation. / In a different setup, similar column were packed with a PCB contaminated soil. These soil columns were bioaugmented with three bacterial cultures, i.e., R. meliloti (strain A-025), Comomonas testosteroni (strain B-356) and an indigenous bacterial consortium using subirrigation. The results indicated that bioaugmentation of the PCB contaminated soil was possible by using subirrigation. Bioaugmentation with the indigenous culture was observed to be more effective in the biodegradation of PCBs than with A-025 and B-356 cultures at 140 and 340 mm depths. However, at 590 mm depth, bioaugmentation with strain A-025 was observed to be better than the other treatments. Sequential aerobic and anaerobic cycles appear to be of significance for effective dechlorination of PCB congeners to lower chlorinated congeners. / In a separate exploratory study, the rhizospheric effects of alfalfa plants on R. meliloti for PCB depletion were investigated. The results suggest that the growth of alfalfa plants and bioaugmentation of soil with R. meliloti, strain A-025, increased the depletion of PCB congeners in the soil as compared to bioaugmentation alone. In other preliminary studies, the results showed that the presence of PCBs in a sandy loam soil increases the filtration of bacterial cells. Also, soil type and the presence of PCBs affected water infiltration, moisture, and hardness of the soil. Furthermore, water table management system along with bioaugmentation of soil columns with R. meliloti, strain A-025, decreased the concentration of atrazine by 31% during anaerobic and aerobic cycles and reduced the concentration of nitrate by 87% and 78% in the absence and presence of atrazine, respectively, in the drainage water. / The overall results of this work indicate that water table management (subirrigation) can be used for bioaugmentation of contaminated soils. Also, use of R. meliloti may prove to be an interesting option for soils contaminated with PCBs, atrazine and nitrate.

The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.

In the N2 fixing legume symbiosis, mineral nitrogen (N) not only decreases N2 fixation, but also delays and inhibits the formation and development of nodules. The purposes of this thesis were to elucidate the role of signaling in the mineral N effects on nodulation and nitrogen fixation in soybean [Glycine max (L.) Merr.] and to attempt to find ways to overcome this inhibition. The responses of soybean plants, in terms of daidzein and genistein synthesis and exudation, to different mineral N levels were studied. Daidzein and genistein distribution patterns varied with plant organs, mineral N levels, and plant development stages. Mineral N inhibited daidzein and genistein contents and concentrations in soybean root and shoot extracts, but did not affect root daidzein and genistein excretion in the same way. In both synthesis and excretion, daidzein and genistein were not affected equally by mineral N treatments. Variability existed among soybean cultivars in the responses of root daidzein and genistein contents and concentrations to mineral N levels. The amount of daidzein and genistein excreted by soybean roots did not always correspond to the daidzein and genistein contents and concentrations inside the roots. On the Bradyrhizobium japonicum side, nod gene expression was inhibited by mineral nitrogen. Genistein was used to pre-incubate B. japonicum cells or was applied directly into the plant growing medium. The results showed that genistein manipulation increased nodule weight and nodule nitrogen fixation under greenhouse conditions, but interactions existed among soybean cultivars, genistein concentrations and nitrate levels. Similar results were found under field conditions. Soybean yield was increased on sandy-loam soil by preincubation of B. japonicum cells with genistein. Addition of genistein beginning at the onset of nitrogen fixation also improved soybean nodulation and nitrogen fixation. Soybean cultivars had different sensitivities to genistein additi / Other studies also show that temperature affected genistein and daidzein content and concentration in soybean roots. The effect of temperature varied among soybean cultivars. Some PGPR strains can mitigate the negative effects of nitrate on soybean nodulation and nitrogen fixation, however, this is influenced by soybean genotype. Applying PGPR together with genistein preincubation of B. japonicum cells improved soybean nodulation and increased yield. The level of improvement varied among soybean cultivars and PGPR strains. Preincubation of B. japonicum cells with genistein improved strain competitiveness under greenhouse, but not field conditions. / Overall, these findings suggested that both plant-to-Bradyrhizobium and Bradyrhizobium-to-plant signals play important roles in the effects of mineral N on nodulation and N fixation. Signal manipulation could partially overcome the inhibitory effects of mineral N on soybean- B. japonicum N fixation symbiosis.

In this study, lipo-chitooligosaccharide (NodBj-V (C 18:1, MeFuc); LCO) 10-7M, extracted from Bradyrhizobium japonicum, was sprayed on the leaves of soybean cv. OAC Bayfield soybean and Evans x L66-2470 (carrying the rj1 mutation, and unable to nodulate). Leaf SA level and activities of the PR proteins chitinase, beta-1,3-glucanase and guaiacol peroxidase (GPOX) were quantified. Phenylalanine ammonia-lyase 1 (PAL1) and isoflavone synthase 2 (IFS2) relative gene expression levels in the sprayed leaves were quantified using quantitative real-time PCR. Messenger RNA abundance was quantified using microarrays. The treatment caused a transient increase in local salicylate levels 24 h after exposure, and a systemic increase in GPOX activity 48 h after exposure, in both soybean types. Of the selected 38 genes affected by the LCO treatment, 25 were stress-related. There were no significant differences in (A) chitinase and beta-1,3-glucanase activity, or (B) in PAL1 and IFS2 gene expression.

Soybean (Glycine max L. Merr.) and Bradyrhizobium japonicum can form a nitrogen fixing symbiosis. This symbiosis is important for most sustainable agriculture systems. This thesis examines two ways to enhance nodulation and nitrogen fixation by this symbiosis: coinoculation of plant growth promoting bacteria (PGPB) with B. japonicum, and addition of RNA to a bradyrhizobial culture medium. The optimal coinoculation dose of Serratia proteamaculans 1--102 and S. liquefaciens 2--68 was determined as 108 cells per plant under both optimal and suboptimal root zone temperatures (RZTs). Nodulation dynamics studies indicated that coinoculation of these two PGPB caused earlier nodule initiation and a higher nodulation rate, contributing to the higher nodule number and nodule weight. The coinoculation also increased nitrogen fixation efficiency under both optimal and suboptimal RZTs. A novel inducible activator only produced by the bacteria after addition of flavonoids to the culture system was prepared and evaluated in greenhouse and field experiments. Fourteen non-bradyrhizobial endophytic bacteria (NEB) were isolated from the surface sterilized root nodules, and three of these, designated NEB4, NEW and NEB17, showed soybean plant growth promotion under both greenhouse (with controlled RZTs) and field conditions. Alone, they were neither nodule inducers nor nitrogen fixers. Biolog tests and partial 16S rRNA gene sequence analyses placed the three strains in genus Bacillus: NEB4 and NEB5 are B. subtilis and NEB17 B. thuringiensis. Bradyrhizobium species grow slowly, making the culture process long and the cost of inoculant production higher. Addition of commercial yeast RNA to the bacterial culture medium accelerated the bacterial growth rate, shortened the culture time and increased the lipo-chitooligosaccharide (LCO) yield in flask cultures. Inoculation experiments in the greenhouse also showed that bradyrhizobial inoculant produced in the presence of RNA had better symb

Plants possess highly sensitive perception systems by which they recognize signal compounds originating from microbes. These molecular cues play an important role in both symbiotic and pathogenic relationships. Establishment of the soybean (Glycine max)-Bradyrhizobium symbiosis is orchestrated by specific signal molecules exchanged between appropriate plant and microbe partners: flavonoids as plant-to-bacteria signals, and Nod factor as bacteria-to-plant signals. How this signaling process interacts with stress conditions (abiotic and biotic) is the subject of this thesis. The abiotic stresses were suboptimal growth temperature, low pH, and salinity. Suboptimal growth temperatures affected the ability of the microsymbiont, Bradyrhizobim japonicum, to perceive nod gene inducers (genistein) and produce Nod factor. Nod Bj-V (C18:1, MeFuc) production by B. japonicum strains 523C and USDA110 was strongly affected by suboptimal growth temperature. Nod factor production declined with temperature, from 28 to 15°C. Strain USDA110 was more affected by decreased temperature than strain 532C. Decreased Nod factor production at low temperature was due to both decreased bacterial growth and lower production efficiency (Nod factor per cell). When a 1:1 mixture of Nod factor Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc) was applied to soybean roots, root hair deformation increased as Nod factor concentration increased under stressfully low temperature and low pH conditions. High salinity stress strongly reduced the root hair deformation caused by Nod factor, and increasing the concentrations of added Nod factor did not over come this. Exogenous application of Nod Bj-V (C18:1, MeFuc), from strain 532C, to soybean root systems under two root zone temperatures (RZTs---17 and 25°C) reduced the progression of disease (powdery mildew---Microsphaera difussa) development on soybean leaves; this effect increased with Nod factor concentration and was gr

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.

Perennial clovers are being evaluated for their potential to reduce groundwater levels in Australian cropping zones where many soils are considered too acidic for reliable lucerne nodulation. However, the release of effective inocula for perennial clovers into such areas where sub clover is the predominant legume, could potentially compromise nitrogen fixation from this valuable annual clover if the symbiosis between the new inoculants and sub clover is not optimal. Studies were therefore designed to increase our understanding of these symbiotic interactions to optimise the management of legume-rhizobia interactions to extend (rather than restrict) the use of legumes in new environments. To assist the understanding of interactions between clovers and their microsymbionts, a glasshouse-based study of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum biovar trifolii (R. l. trifolii) associated with 38 genotypes of annual and perennial Trifolium spp. from world centres of diversity was undertaken. Rhizobial isolates and clovers were assembled from South and equatorial Africa, North and South America and the Euro-Mediterranean regions. There was substantial specificity amongst the African clovers for effective nodulation. No strain of rhizobia from the South American perennial T. polymorphum, or from the Ethiopian clovers, was able to nodulate sub clover effectively, whilst less than 33% of the 18 strains from these regions could form nodules with the less promiscuous Mediterranean annual T. glanduliferum. Seventy of 476 cross-inoculation treatments examined did not nodulate, whilst 81 treatments clearly demonstrated effective nodulation. The remainder of the crossinoculation pairings revealed only partially effective or ineffective nodulation. Two barriers to effective nodulation were identified from the cross- inoculation study: a geographic barrier representing the broad centres of clover diversity, across which few host- strain combinations were effective; and within each region, a significant barrier to effective nodulation between an isolate from an annual host on a perennial host, or vice versa. Clovers and their rhizobia from within the Euro-Mediterranean region of diversity were more able to overlap the annual/perennial barrier than genotypes from the other regions. The data indicate that it will be a substantial challenge to develop inocula for perennial clovers that do not adversely affect nitrogen fixation by sub clover and other annual clovers in commerce, especially if the perennial clovers originate from Africa or America. To investigate the management of legume-rhizobia interactions when introducing legumes into new environments, a study was initiated in Uruguay (Mediterranean annual clovers were introduced into a predominantly perennial clover setting) that could be considered opposite to the situation emerging within southern Australia (perennial clovers evaluated in a predominantly annual clover setting). The Uruguayan grasslands contain populations of indigenous R. l. trifolii that nodulate endemic T. polymorphum but form ineffective nodules on clovers originating from the Mediterranean region. Importantly in the Uruguayan setting, Government policy has facilitated the introduction of numerous varieties of annual Mediterranean clovers with the aim of improving overall winter production in their naturally managed grasslands. In an attempt to understand the rhizobial ecology of this scenario, a cross-row experiment was set-up in 1999 in a basaltic, acid soil in Glencoe, Uruguay, to follow the survival and symbiotic performance of nine exotic strains of R. l. trifolii. In this thesis I report on the ability of the introduced strains to compete for nodule occupancy of Mediterranean clover hosts and show the impacts of the introduced strains on the productivity of the indigenous Uruguayan clover, T. polymorphum. Of the introduced strains, WSM1325 was a superior inoculant and remained highly persistent and competitive in forming effective symbioses with the Mediterranean hosts, T. purpureum and T. repens, in the Uruguayan environment over a 3 year period. T. purpureum and T. repens, when inoculated with the introduced strains, did not nodulate with any indigenous R. l. trifolii as typed from nodules of T. polymorphum. Conversely, there were no nodules on the Uruguayan host T. polymorphum that contained the introduced R. l. trifolii. These results revealed that there were effective symbioses between strains of R. l. trifolii and clovers, even though the soil contained ineffective R. l. trifolii for all hosts. This represents the first reported example of selective nodulation for an effective symbiosis in situ with annual and perennial clovers in acid soils. This phenomenon raised the question of whether this was restricted to the particular edaphic scenario in Glencoe, Uruguay. Glasshouse-based experiments in Australia were conducted to further understand the selection phenomenon. Two strains were selected for comparisons; strain WSM1325 isolated from an annual clover in the Mediterranean and WSM2304 isolated from the perennial clover T. polymorphum in Uruguay, South America. Variables that may have been specific to Glencoe were investigated. Thus, the effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were examined on the two hosts (T. purpureum and T. polymorphum). Each was exposed to the same effective and ineffective micro-symbionts. In co-inoculation experiments at a cell density of 104 cells mL-1, each host nodulated solely with its effective strain, even when this strain was out-numbered 100:1 by the ineffective strain. However, the selection process ceased when the effective strain was out-numbered 1000:1. At higher basal cell concentrations of 105 - 108 cells mL-1, selection for WSM1325 to form effective nodules on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. These results indicate that the selection mechanism is highly dependent upon the basal rhizobial cell density. Soil pH did not significantly alter the process, which could not be simply explained by the rate of strain growth, or extent of nodulation. Greater precision was sought in the terminology applied to nodulation outcomes where legumes have a choice of micro-symbiotic partners from within the same species of root-nodule bacteria. The nominated preferred terms are “nonselective”, “exclusive”, and “selective” nodulation. In view of the difference in host range between WSM1325 and WSM2304 and the selective nodulation process, a preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 was conducted. A selected range of gene regions were amplified by PCR from each strain and sequenced. Comparative analysis of the nucleotide sequences revealed that although the 16S rRNA sequences were identical, the atpD, GSII and nodD sequences contained distinct differences revealing disparity between the pSym replicons and between the chromosomal replicons of these strains. Of the genes sequenced, the highest degree of divergence was noted for the symbiotic NodD protein products, which are known to be critical determinants in the nodulation of specific hosts. An examination of the nodD gene region of WSM1325 and WSM2304 revealed a further contrasting feature; the regulatory gene nodR was present in the nodD gene region of WSM1325 but absent in WSM2304. Since NodR is known to be required for adding highly unsaturated fatty acyl groups onto the Nod-factor backbone, I could now hypothesise that the nodulation incompatibility observed between Trifolium hosts and micro-symbionts obtained from different geographical locations may result from differences in Nod-factor decoration. With the full genome sequence of the two strains WSM1325 and WSM2304 soon to be available, the role of nodR and any link to the selection phenomenon described in this thesis can be addressed.