The two-component colR/S system of Pseudomonas fluorescens WCS365 plays a role in rhizosphere competence through maintaining the structure and function of the outer membrane

Pseudomonas fluorescens strain PCL1210, a competitive tomato root tip colonization mutant of the efficient root colonizing wild type strain WCS365, is impaired in the two-component sensor-response regulator system ColR/ColS. Here we show that a putative methyltransferase/wapQ operon is located downstream of colR/colS and that this operon is regulated by ColR/ColS. Since wapQ encodes a putative lipopolysaccharide (LPS) phosphatase, the possibility was studied that the integrity of the outer membrane of PCL1210 was altered. Indeed, it was shown that mutant PCL1210 is more resistant to various chemically unrelated antibiotics which have to pass the outer membrane for their action. In contrast, the mutant is more sensitive to the LPS-binding antibiotic polymyxin B. Mutant PCL1210 loses growth in competition with its wild type when grown in tomato root exudate. Mutants in the methyltransferase/wapQ operon are also altered in their outer membrane permeability and are defective in competitive tomato root tip colonization. A model for the altered outer membrane of PCL1210 is discussed.     

Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.     

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.     

Increased uptake of putrescine in the rhizosphere inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.     

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.     

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.     

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.     

Biocontrol strain Pseudomonas fluorescens WCS365 inhibits germination of Fusarium oxysporum spores in tomato root exudate as well as subsequent formation of new spores

Fusarium oxysporum f.sp. radicis-licopersici (Forl) is a soilborne pathogenic fungus which can cause tomato foot and root rot (TFRR). Tomato root exudate is a good source of nutrients for both Forl and the TFRR-suppressing biocontrol bacterium Pseudomonas fluorescens strain WCS365. Incubation of Forl microconidia in tomato root exudate stimulates their germination. This phenomenon is observed, to a lesser extent, upon incubation in plant nutrient solution supplemented with citrate or glucose, the major organic acid and sugar components, respectively, of tomato root exudate. Here we show that induction of germination of microconidia is significantly reduced in the presence of P. fluorescens WCS365 in all tested media. Scanning electron microscopy revealed that P. fluorescens WCS365 colonizes developing hyphae. Efficient colonization correlates with low nutrient availability. Eventually, new microconidia are formed. The presence of P. fluorescens WCS365 reduces the number of newly formed microconidia. This reduction does not depend on physical contact between bacteria and hyphae. We discuss that the ability of P. fluorescens WCS365 to slow down the processes of microconidia germination and development of new microconidia of the phytopathogen, and therefore the ability to reduce fungal dissemination, is likely to contribute to the biocontrol efficacy of this strain.     

Generation of enhanced competitive root-tip-colonizing Pseudomonas bacteria through accelerated evolution

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A. G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.     

Collimonas fungivorans, an unpredicted in vitro but efficient in vivo biocontrol agent for the suppression of tomato foot and root rot

Although bacteria from the genus Collimonas have demonstrated in vitro antifungal activity against many different fungi, they appeared inactive against the plant-pathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici (Forl), the causal agent of tomato foot and root rot (TFRR). Visualization studies using fluorescently labelled organisms showed that bacterial cells attached extensively to the fungal hyphae under nutrient-poor conditions but not in glucose-rich Armstrong medium. Collimonas fungivorans was shown to be as efficient in colonizing tomato root tips as the excellent colonizer Pseudomonas fluorescens strain WCS365. Furthermore, it appeared to colonize the same sites on the root as did the phytopathogenic fungus. Under greenhouse conditions in potting soil, C. fungivorans performed as well in biocontrol of TFRR as the well-established biocontrol strains P. fluorescens WCS365 and Pseudomonas chlororaphis PCL1391. Moreover, under biocontrol conditions, C. fungivorans did not attach to Forl hyphae colonizing plant roots. Based on these observations, we hypothesize that C. fungivorans mainly controls TFRR through a mechanism of competition for nutrients and niches rather than through its reported mycophagous properties, for which attachment of the bacteria to the fungal hyphae is assumed to be important.     

Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector.     

Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.     

Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365 on the composition of organic acids and sugars in tomato root exudate

The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate revealed that the presence of F. oxysporum f. sp. radicis-lycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased, mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.     

Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate

AIMS: Tomato foot and root rot (TFRR), caused by Fusariumoxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root-colonizing bacteria from total rhizosphere soil samples. METHODS AND RESULTS: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains. CONCLUSIONS: The new biocontrol strains, six Gram-negative and one Gram-positive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.     

Enrichment for enhanced competitive plant root tip colonizers selects for a new class of biocontrol bacteria

Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl (Fusarium oxysporum f.sp. radicis-lycopersici ). Several bacteria have been described to be able to control the disease, using different mechanisms. Here we describe a method that enables us to select, after application of a crude rhizobacterial mixture on a sterile seedling, those strains that reach the root tip faster than our best tomato root colonizer tested so far, the Pseudomonas fluorescens biocontrol strain WCS365. Of the five tested new isolates, four appeared to be able to reduce the number of diseased plants. Analysis of one of these strains, P. fluorescens PCL1751, suggests that it controls the disease through the mechanism 'competition for nutrients and niches', a mechanism novel for biocontrol bacteria. Moreover, this is the first report describing a method to enrich for biocontrol strains from a crude mixture of rhizobacteria. Another advantage of the method is that four out of five strains do not produce antifungal metabolites, which is preferential for registration as a commercial product.     

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.     

Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.     

Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli.

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.     

Mutagenesis of subunit N of the Escherichia coli complex I. Identification of the initiation codon and the sensitivity of mutants to decylubiquinone

The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.     

Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots.

The role of motility in the colonization of potato roots by Pseudomonas bacteria was studied. Four Tn5-induced flagella-less mutants of the plant-growth-stimulating P. fluorescens WCS374 appeared to be impaired in their ability to colonize growing potato roots.