RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.     

Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport

The genes controlling the transport of C4-dicarboxylic acids from Rhizobium meliloti have been cloned and analysed. The nucleotide sequence of the control region of the structural dctA and the regulatory dctBD genes has been determined. Comparison with the Rhizobium leguminosarum dct genes revealed a high degree of homology. Gene fusions to the enteric lacZY reporter gene were constructed and the expression of the dctA and dctBD genes studied under various physiological conditions. In free-living cells, the regulatory dctBD genes are absolutely required for the expression of the dctA gene. In the root nodule environment, a dctA::lacZY gene fusion was found to be expressed in an R. meliloti strain mutated in both the dctB and dctD genes, but not in a strain mutated in the dctB gene alone. The presence of the conserved upstream NifA-binding sites on the dctA promoter sequence, coupled with the fact that the dctA::lacZY gene fusion is not expressed in root nodules formed by a nifA mutant strain of R. meliloti, supports the suggestion that NifA may be involved in the symbiotic expression of dctA in the absence of the regulatory dctBD genes. Under micro-aerobic conditions, however, NifA induction alone is not sufficient for expression of the dctA promoter, even though the NifA-dependent nifHDK promoter is highly expressed under these conditions.     

Phenazine-1-carboxylic acid biosynthesis in <Emphasis Type="Italic">Pseudomonas Chlororaphis</Emphasis> GP72 is positively regulated by the sigma factor RpoN

The production of phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) makes Pseudomonas chlororaphis GP72 an effective biocontrol agent. In order to understand how production of PCA is regulated by RpoN, an insertional mutation in rpoN has been made in P. chlororaphis GP72. Production of PCA in the rpoN mutant strain GP72N decreased both in King’s B medium and in Pigment Producing Medium. Moreover, the expression of the translational fusion phzA′–′lacZ was reduced about 2-fold in GP72N compared to wild type strain, whatever the growth medium is. Complementation of rpoN gene in mutant GP72N restored its motility and its PCA biosynthesis ability. However, overexpression of RpoN had no major effects on the expression of the RpoN-dependent phenotypes described in this study for P. chlororaphis GP72. These results suggest that RpoN is involved as a positive regulator in the regulation of PCA biosynthesis in P. chlororaphis GP72.     

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.     

Root exudates of wetland plants influenced by nutrient status and types of plant cultivation

The present study investigated the amounts of root exudates and composition of organic acids released from two wetland plants (Typha latifolia and Vetiver zizanioides) under two nutrient treatments: low level (0.786 mM N and 0.032 mM P) and high level (7.86 mM N and 0.32 mM P) and two types of plant cultivation: monoculture and co-culture of the two plants. Low nutrient treatment significantly (p < 0.05) increased the root exudates of T. latifolia during the initial growth period (1-21 d) and those of V. zizanioides and the co-culture during the whole growth period. The concentrations of dissolved organic carbon in the root exudates of the co-culture in the low nutrient treatment were 3.23-7.91 times of those in the high nutrient treatment during the medium growth period (7-28 d). The compositions of organic acids varied between the two plant species and between the two nutrient treatments. The pattern of organic acids was also different between the co-culture and the monoculture. Oxalic acid was by far the major organic acid exuded from the two wetland plants. The present study on root exudates suggests that co-culture of wetland plant species would be more useful in the reclamation of waste water than a monoculture system.     

富钾基因型籽粒苋主要根系分泌物及其对土壤矿物态钾的活化作用

通过试验,研究了2种供K水平对籽粒苋(Amaranthus spp.)富K基因型和一般基因型根系分泌物含量变化的影响,以及在低K胁迫时3个生长期两类基因型主要根系分泌物含量的变化特点,模拟了籽粒苋根系分泌物对土壤矿物态钾的活化作用.结果表明,籽粒苋根系分泌物中可溶性糖、氨基酸和有机酸含量随供K水平的升高而降低,且富K基因型根系分泌物中3种物质的分泌量始终大于一般基因型;在正常供K条件下,两基因型根系分泌能力相近,但在低K处理时,前者显著高于后者,差异显著;在2种供K水平下,根系有机酸分泌量在3种分泌物中占绝对优势,分别是可溶性糖和氨基酸分泌量的几十倍和几百倍.籽粒苋生长到50 d时,一般基因型根系可溶性糖、氨基酸和有机酸的分泌量较40 d时迅速降低.富K基因型根系分泌物中可溶性糖、氨基酸和有机酸含量在3个生长时期均大于一般基因型,且随着生长时间的延长,两基因型间可溶性糖、氨基酸和有机酸含量的差异明显增大.两类基因型在3个生长时期均以分泌有机酸为主,其占总分泌量的93%以上.籽粒苋根系分泌物处理后的土壤速效钾含量均高于清水对照处理,富K基因型在低K胁迫时的根系分泌物对土壤K的活化作用明显大于一般基因型.     

芘对黑麦草根系几种低分子量有机分泌物的影响

植物根系释放分泌物与有机污染物的植物修复机制密切相关,研究具有有机污染物修复潜力植物在污染胁迫条件下的根系分泌物特征有助于揭示其修复机制.以多环芳烃修复研究中常用的黑麦草为材料(Lolium perenneL.)为材料,在营养液栽培方式下研究了在芘胁迫处理下,黑麦草根系几种低分子量有机物分泌特征.结果表明,黑麦草对芘具有较强的耐受能力,芘胁迫处理下,生物量无显著变化.黑麦草根系分泌的低分子量有机酸主要为草酸.3、6 mg/L和9 mg/L芘胁迫处理下,低分子量有机酸的组成无明显变化,但含量随芘胁迫处理浓度上升而显著增加(P＜0.05);总糖分泌量随着芘胁迫处理浓度升高而呈现先略微上升后下降的趋势,相对高峰值出现在芘胁迫处理浓度3 mg/L,但差异不显著;氨基酸分泌总量随着芘胁迫处理浓度的增加而显著增多,芘胁迫浓度在3、6 mg/L和9 mg/L时,根系氨基酸的分泌总量分别是空白的1.37、2.02倍和2.65倍,但根系分泌的氨基酸组成无明显变化,19种常见氨基酸分泌的数量变化情况却不相同,分泌量总体均随着芘胁迫处理浓度的提高而增加,其中苏氨酸、丝氨酸、脯氨酸、甘氨酸、丙氨酸、亮氨酸、组氨酸和鸟氨酸的分泌量显著增多,差异显著(P＜0.05).     

连作大豆根分泌物对根腐病病原菌的化感作用

采用砂培,水培和室内培养等试验方法研究了连作大豆根分泌物对根腐病病原菌的化感作用。结果表明,与对照相比,连作和轮作大豆根分泌物对半镰孢菌,粉红粘帚功和尖镰孢菌尤其是对半裸镰孢菌的生长有明显的化感促进作用,差异达显著或极显著水平,低浓度时,连作大豆根分泌物对半裸镰孢菌和粉红粘帚菌生长的化感促进作用明显大于轮作大豆,差异达显著水平,同一茬口,高浓度根分泌物半裸镰孢菌生长的化感促进作用小于低浓度,而且在连作大豆中差异达显著水平。与对照相比,高浓度的邻苯二甲酸和丙二酸（L5和B5）对半裸镰孢菌,粉红粘帚菌和尖镰孢菌尤其是对半裸镰孢菌的生长有化感抑制作用,差异达显著或极显著水平,而低浓诬的邻苯二甲酸和丙二酸对半裸镰孢菌,粉红粘帚菌和尖镰孢菌的生长有化感促进,部分差异达显著水平。     

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.     

Plant growth strategy determines the magnitude and direction of drought-induced changes in root exudates in subtropical forests

Root exudates are an important pathway for plant–microbial interactions and are highly sensitive to climate change. However, how extreme drought affects root exudates and the main components, as well as species-specific differences in response magnitude and direction, are poorly understood. In this study, root exudation rates of total carbon (C) and its components (e.g., sugar, organic acid, and amino acid) were measured under the control and extreme drought treatments (i.e., 70% throughfall reduction) by in situ collection of four tree species with different growth rates in a subtropical forest. We also quantified soil properties, root morphological traits, and mycorrhizal infection rates to examine the driving factors underlying variations in root exudation. Our results showed that extreme drought significantly decreased root exudation rates of total C, sugar, and amino acid by 17.8%, 30.8%, and 35.0%, respectively, but increased root exudation rate of organic acid by 38.6%, which were largely associated with drought-induced changes in tree growth rates, root morphological traits, and mycorrhizal infection rates. Specifically, trees with relatively high growth rates were more responsive to drought for root exudation rates compared with those with relatively low growth rates, which were closely related to root morphological traits and mycorrhizal infection rates. These findings highlight the importance of plant growth strategy in mediating drought-induced changes in root exudation rates. The coordinations among root exudation rates, root morphological traits, and mycorrhizal symbioses in response to drought could be incorporated into land surface models to improve the prediction of climate change impacts on rhizosphere C dynamics in forest ecosystems.     

Aromatic compounds degradation plays a role in colonization of Arabidopsis thaliana and Acacia caven by Cupriavidus pinatubonensis JMP134

Plant rhizosphere and internal tissues may constitute a relevant habitat for soil bacteria displaying high catabolic versatility towards xenobiotic aromatic compounds. Root exudates contain various molecules that are structurally related to aromatic xenobiotics and have been shown to stimulate bacterial degradation of aromatic pollutants in the rhizosphere. The ability to degrade specific aromatic components of root exudates could thus provide versatile catabolic bacteria with an advantage for rhizosphere colonization and growth. In this work, Cupriavidus pinatubonensis JMP134, a well-known aromatic compound degrader (including the herbicide 2,4-dichlorophenoxyacetate, 2,4-D), was shown to stably colonize Arabidopsis thaliana and Acacia caven plants both at the rhizoplane and endorhizosphere levels and to use root exudates as a sole carbon and energy source. No deleterious effects were detected on these colonized plants. When a toxic concentration of 2,4-D was applied to colonized A. caven, a marked resistance was induced in the plant, showing that strain JMP134 was both metabolically active and potentially beneficial to its host. The role for the β-ketoadipate aromatic degradation pathway during plant root colonization by C. pinatubonensis JMP134 was investigated by gene inactivation. A C. pinatubonensis mutant derivative strain displayed a reduced ability to catabolise root exudates isolated from either plant host. In this mutant strain, a lower competence in the rhizosphere of A. caven was also shown, both in gnotobiotic in vitro cultures and in plant/soil microcosms.     

An allele of gyrA prevents Salmonella enterica serovar Typhimurium from using succinate as a carbon source

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C(4)-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C(4)-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.     

OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice.