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Steven E Lindow

Publications and source records attributed to Steven E Lindow.

15 recordsLinked to original sources
PubMed2006

Disruption of N-acyl homoserine lactone-mediated cell signaling and iron acquisition in epiphytic bacteria by leaf surface compounds.

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C8 and C18 reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.

4-Butyrolactone↗
PubMed2005

Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000.

The complete genomic sequence of Pseudomonas syringae pv. syringae B728a (Pss B728a) has been determined and is compared with that of P. syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 Mb) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. Although a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared with Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences unique to Pss B728a when compared with Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands varies, with one containing a prophage and another the plasmid pKLC102 of Pseudomonas aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin, syringomycin, indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to the epiphytic fitness and stress tolerance of this organism.

Bacterial Proteins↗
PubMed2005

Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae.

The N-acyl homoserine lactone (AHL)-mediated quorum-sensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P syringae were examined. Both an aefR- mutant and an ahlI- ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in E syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.

Fabaceae↗
PubMed2005

Pseudomonas syringae genes induced during colonization of leaf surfaces.

The foliar pathogen and ice nucleator, Pseudomonas syringae pv. syringae B728a, demonstrates a high level of epiphytic fitness on plants. Using a promoter-trapping strategy termed habitat-inducible rescue of survival (HIRS), we identified genes of this organism that are induced during colonization of healthy bean leaf surfaces. These plant-inducible genes (pigs) encode diverse cellular functions including virulence, transcription regulation, transport, nutrient acquisition and other known and unknown loci, some of which may result in antisense transcripts to annotated P. syringae genes. Prominent among the pigs was ssuE, a gene in the sulfate-starvation regulon, indicating that sulfate is not abundant on leaf surfaces. inaZ reporter gene fusion assays of the plant-inducible loci revealed up to 300-fold higher levels of pig transcriptional activity on plant leaves compared with minimal medium. However, the maximum levels of pig transcriptional activity were typically too weak to be measured using a gfp reporter gene. One exception was orf6 in the hrp/hrc pathogenicity island which was highly induced in epiphytic P. syringae cells. Four pigs were disrupted by insertional mutagenesis. While growth of the ssuE mutant was impaired under certain conditions in laboratory medium, the epiphytic and virulence properties of the mutants on bean plants were identical to wild-type P. syringae. Our results demonstrate the utility of HIRS to identify genes expressed on leaves and provide new insight into the leaf surface environment.

Amino Acid Sequence↗
PubMed2005

Two novel bacterial biosensors for detection of nitrate availability in the rhizosphere.

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5alpha and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 microM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 microM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.

Aerobiosis↗
PubMed2005

Utilization of the plant hormone indole-3-acetic acid for growth by Pseudomonas putida strain 1290.

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.

Base Sequence↗
PubMed2004

Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants.

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect.

Amino Acid Sequence↗
PubMed2004

Regulation of AHL production and its contribution to epiphytic fitness in Pseudomonas syringae.

Pseudomonas syringae forms large cell aggregates that are more stress tolerant than solitary cells during epiphytic growth on plants. The differential survival of aggregates on leaves suggests that epiphytic fitness traits may be controlled in a density-dependent manner via cell-cell signaling. We investigated this hypothesis in P. syringae B728a. Synthesis of N-acyl-homoserine lactone (AHL), 3-oxo-hexanoyl homoserine lactone, and the expression of the gene encoding AHL synthase ahlI were maximal at high cell concentrations. The expression of the AHL regulator ahlR, in contrast, was similar at all cell concentrations. A screen of Tn5 mutants revealed that P. syringae B728a requires a novel transcriptional activator for AHL production. This regulator, which belongs to the TetR family, was also required for epiphytic fitness and has been designated AefR (for AHL and epiphytic fitness regulator). The expression of ahlI was greatly reduced in both aefR- and gacA- mutants and was completely restored in either mutant after addition of exogenous AHL. In contrast, the expression of aefR was not reduced in either gacS- or gacA- mutants. Thus, AefR appears to positively regulate AHL production independently of the regulators GacS/GacA and also controls traits in P. syringae B728a that are required for epiphytic colonization.

4-Butyrolactone↗
PubMed2004

Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array.

A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT::Tn5), and an eutR homolog from Salmonella typhimurium (eutR::TnS) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.

Base Sequence↗
PubMed2003

Conditional survival as a selection strategy to identify plant-inducible genes of Pseudomonas syringae.

A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of Pseudomonas syringae that are induced during growth on bean leaves. This strategy is based on the complementation of metXW, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of P. syringae on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type P. syringae metXW genes. Only with an active promoter fused to metXW on pTrap did this plasmid restore methionine prototrophy to the P. syringae metXW mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of P. syringae genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique RsaI restriction pattern of their DNA insert. Sequence analysis of these clones revealed those P. syringae genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a gfp reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many P. syringae genes have relatively low expression levels and that the metXW HIRS strategy is a sensitive method to detect weakly expressed P. syringae genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for P. syringae expressing metXW than exposed sites. Thus, the metXW HIRS strategy should lead to the identification of P. syringae genes that are expressed primarily in these areas on the leaf.

Acetyltransferases↗
PubMed2003

Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera.

Xylella fastidiosa causes Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Green Fluorescent Proteins↗
PubMed2002

Phyllosphere microbiology.

Aerial plant surfaces harbor large numbers of microbes, some of which are deleterious to plants whereas others are benign or beneficial. Commercial formulations of bacteria antagonistic to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage. Molecular genetic tools, microscopic examination and whole-cell bacterial biosensors have provided extensive information on these microbes, their complex associations and their habitat. The aerial habitat influenced by plants, termed the phyllosphere, is particularly amenable to studies of microbial ecology and the information gained should lead to more effective means of plant protection.

Bacteria↗
PubMed2002

Bioreporters in microbial ecology.

Bioreporters are effective research tools for gaining an understanding of a microbe's perception of the world. Fitted with a fusion of an environmentally responsive promoter to a suitable reporter gene, a bacterial or fungal bioreporter is able to communicate its metabolic or transcriptional behavior in a habitat, and furnish us with information on the chemical, physical or biological properties of its immediate surroundings. This review details recent developments in the use of such bioreporters in microbial ecology. Emphasis is placed on reporter genes that allow detection in individual microbial cells, as they provide a high-resolution description of the habitat under investigation. In an outlook on the future of bioreporter technology, this review stresses the need to interpret the activity of a bioreporter within the context of its biology.

Bacteria↗
PubMed2000

Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor.

Ferric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low. Thus it is a limiting resource for which microbes must compete in natural habitats. Since competition for iron occurs at the level of individual cells, knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats. Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy. Cells of this iron biosensor exhibited iron-dependent GFP fluorescence that was inversely proportional to the amount of iron added to the media, and which differed by over 20-fold in iron-replete compared to iron-deplete culture media. Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities. In contrast, the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly. The distribution of fluorescence intensities was strongly right-hand skewed, with about 10% of the cells exhibiting substantially higher GFP fluorescence than that of the median cell. Cells of a positive control strain, harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene, exhibited uniform GFP fluorescence both in culture media and on plants. These results indicate that there is substantial heterogeneity of iron biovailability to cells of P. syringae on plants, with only a small subset of cells experiencing low iron availability. Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.

Biological Availability↗