Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.     

Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens

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Weak rolling adhesion enhances bacterial surface colonization

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as Escherichia coli when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.     

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.     

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis

The processes leading to bacterial colonization on solidwater interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 mum (for silicon) to 0.015 mum (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varried by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.     

Inhibition of the bacterial surface protein anchoring transpeptidase sortase by medicinal plants

Inhibition by medicinal plant extracts of a recombinant sortase was evaluated for antibacterial drug discovery. The coding region of sortase, a transpeptidase that cleaves surface proteins of gram-positive bacteria, was amplified by PCR from the chromosome of Staphylococcus aureus ATCC 6538p with the exception of an N-terminal membrane anchor sequence, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The purified sortase had maximum activity at pH 7.5 and was stable at 20-45 degrees C for the cleavage of a synthetic fluorophore substrate. The enzyme inhibitory activity in medicinal plants was also evaluated for antibacterial drug discovery. Among 80 medicinal plants tested, Cocculus trilobus, Fritillaria verticillata, Liriope platyphylla, and Rhus verniciflua had strong inhibitory activity. The extract with the greatest activity was the ethyl acetate fraction derived from the rhizome of Cocculus trilobus (IC50 = 1.52 microg/ml).     

Culturable bacterial communities on leaf sheaths and panicles of rice plants in Japan

Culturable bacterial communities on rice plants were investigated from 2001 to 2003. In total, 1,394 bacterial isolates were obtained from the uppermost leaf sheaths at 1 month before heading time and from leaf sheaths and panicles at heading time. The average culturable bacterial population on the leaf sheaths was larger at heading time than at 1 month previously. Furthermore, the population was significantly larger on panicles than on leaf sheaths, suggesting that the bacterial population is influenced by the organs of rice plants. Larger proportions of bacteria were obtained from the macerates of leaf sheaths after washing with phosphate buffer, and most culturable bacteria were verified to inhabit the inside or inner surface, rather than the outer surface, of the tissues. Verification of the bacterial composition based on 16S rRNA gene sequences revealed that genera of Sphingomonas, Microbacterium, Methylobacterium, and Acidovorax tended to be dominant colonizers on leaf sheaths, whereas Pseudomonas and Pantoea were isolated mainly from the panicles, indicating that leaf sheaths and panicles harbor distinct communities. Furthermore, the richness of bacterial genera was less on both leaf sheaths and panicles at heading time compared with that observed 1 month before heading time. Phylogenetic analyses using bacterial isolates belonging to the four dominant genera inhabiting leaf sheaths at heading time revealed that particular bacterial groups in each genus colonized the leaf sheaths.     

Patterns of leaf surface wetness for montane and subalpine plants

The frequency and duration of water on leaf surfaces have important consequences for plant growth and photosynthetic gas exchange. The objective of the present study was to compare the frequency and duration of leaf wetness under natural field conditions among species and to identify variation in structural features of leaves that may reduce surface wetness. During June–September 1992 in the central Rocky Mountains (USA), natural leaf wetting due to rain and dewfall was observed on 79 of 89 nights in open meadow habitats compared to only 29 of 89 nights in the understorey. Dew formation occurred at relative humidities that were often well below 100% because of radiational heat exchange with cold night skies and low wind speeds (< 0.5 m s^?1). A survey of 50 subalpine/montane species showed that structural characteristics associated with the occurrence and duration of leaf surface wetness differed among species and habitats. Both adaxial and abaxial surfaces accumulated moisture during rain and dewfall events. Leaf surfaces of open-meadow species were less wettable (P= 0.008), and had lower droplet retention (P= 0.015) and more stomata P= 0.017) than adjacent understorey species. Also, leaf trichomes reduced the area of leaf surface covered by moisture. Ecophysiological importance is suggested by the high frequency of leaf wetting events in open microsites, influences on growth and gas exchange, and correspondence between leaf surface wettability and habitat.     

Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple

Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.     

模拟降雨对常绿植物叶表面滞尘的影响

通过模拟降雨实验的方法,在15 mm/h和30 mm/h降雨强度的不同历时条件下,从动态变化、滞尘阈值和建立关系3个方面量化了降雨过程对叶表面不同粒径颗粒物的影响。研究结果表明:叶表面颗粒物滞留率随降雨历时先急剧下降,后趋于稳定状态。降雨初期对叶面尘的影响最为明显,降雨强度较大时洗脱时间更短。颗粒物滞留量和滞留率的阈值均随降雨强度的增加而减小。颗粒物滞留量阈值呈现出10—100μm2.5—10μm0.2—2.5μm的规律,与未降雨前一致。侧柏各粒径颗粒物均能被降雨较有效洗脱;大叶黄杨10—100μm的颗粒物更易被降雨洗脱;油松的颗粒物滞留率阈值达30%—50%,不易被洗脱。降雨量与叶表面颗粒物滞留率有良好的拟合关系,随降雨量的增大,颗粒物滞留率呈指数减小,且减小速率在降雨量10mm内较大,大于10 mm后曲线较为平缓。     

Endophytic root colonization of gramineous plants by Herbaspirillum frisingense

Herbaspirillum frisingense is a diazotrophic betaproteobacterium isolated from C4-energy plants, for example Miscanthus sinensis. To demonstrate endophytic colonization unequivocally, immunological labeling techniques using monospecific polyclonal antibodies against two H. frisingense strains and green fluorescent protein (GFP)-fluorescence tagging were applied. The polyclonal antibodies enabled specific in situ identification and very detailed localization of H. frisingense isolates Mb11 and GSF30(T) within roots of Miscanthusxgiganteus seedlings. Three days after inoculation, cells were found inside root cortex cells and after 7 days they were colonizing the vascular tissue in the central cylinder. GFP-tagged H. frisingense strains could be detected and localized in uncut root material by confocal laser scanning microscopy and were found as endophytes in cortex cells, intercellular spaces and the central cylinder of barley roots. Concerning the production of potential plant effector molecules, H. frisingense strain GSF30(T) tested positive for the production of indole-3-acetic acid, while Mb11 was shown to produce N-acylhomoserine lactones, and both strains were able to utilize 1-aminocyclopropane-1-carboxylate (ACC), providing an indication of the activity of an ACC-deaminase. These results clearly present H. frisingense as a true plant endophyte and, although initial greenhouse experiments did not lead to clear plant growth stimulation, demonstrate the potential of this species for beneficial effects on the growth of crop plants.     

Regulation of enteric endophytic bacterial colonization by plant defenses

Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethylene-insensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. This ethylene-mediated inhibition of endophytic colonization was reversed by addition of the ethylene action inhibitor, 1-methylcyclopropene. Colonization of Medicago spp. by S. typhimurium also was affected by exogenous ethylene. Mutants lacking flagella or a component of the type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1) colonize the interior of Medicago spp. in higher numbers than the wild type. Arabidopsis defense response-related genotypes indicated that only salicylic acid (SA)-independent defense responses contribute to restricting colonization by Kp342. In contrast, colonization by S. typhimurium is affected by both SA-dependent and -independent responses. S. typhimurium mutants further delineated these responses, suggesting that both flagella and TTSS-SPI1 effectors can be recognized. Flagella act primarily through SA-independent responses (compromising SA accumulation still affected colonization in the absence of flagella). Removal of a TTSS-SPI1 effector resulted in hypercolonization regardless of whether the genotype was affected in either SA-dependent or SA-independent responses. Consistent with these results, S. typhimurium activates the promoter of PR1, a SA-dependent pathogenesis-related gene, while S. typhimurium mutants lacking the TTSS-SPI1 failed to activate this promoter. These observations suggest approaches to reduce contamination of raw produce by human enteric pathogens and to increase the number of growth-promoting bacteria in plants.     

Carbon stable isotopic composition of soluble sugars in Tillandsia epiphytes varies in response to shifts in habitat

We studied C stable isotopic composition (δ¹³C) of bulk leaf tissue and extracted sugars of four epiphytic Tillandsia species to investigate flexibility in the use of crassulacean acid metabolism (CAM) and C₃ photosynthetic pathways. Plants growing in two seasonally dry tropical forest reserves in Mexico that differ in annual precipitation were measured during wet and dry seasons, and among secondary, mature, and wetland forest types within each site. Dry season sugars were more enriched in ¹³C than wet season sugars, but there was no seasonal difference in bulk tissues. Bulk tissue δ¹³C differed by species and by forest type, with values from open-canopied wetlands more enriched in ¹³C than mature or secondary forest types. The shifts within forest habitat were related to temporal and spatial changes in vapor pressure deficits (VPD). Modeling results estimate a possible 4% increase in the proportional contribution of the C₃ pathway during the wet season, emphasizing that any seasonal or habitat-mediated variation in photosynthetic pathway appears to be quite moderate and within the range of isotopic effects caused by variation in stomatal conductance during assimilation through the C₃ pathway and environmental variation in VPD. C isotopic analysis of sugars together with bulk leaf tissue offers a useful approach for incorporating short- and long-term measurements of C isotope discrimination during photosynthesis.