Flagella-specific bacteriophages of Agrobacterium tumefaciens: demonstration of virulence of nonmotile mutants

Bacteriophages GS2 and GS6 for Agrobacterium tumefaciens were shown by electron microscopy to adsorb to flagella. This specificity was confirmed by the finding that phage-resistant mutants were nonmotile. Such mutants retained tumor-inducing virulence and ability to attach to plant cells, indicating that motility was not required for these properties. Both phages had contractile tails and appeared similar in the electron microscope.     

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice

Background

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.

Methodology/Principal Findings

The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.

Conclusions/Significance

We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.     

Increased mutation rate of E. coli K12 lambda cultures maintained in continuous logarithmic growth

Continuous logarithmic growth of E. coli K12 lambda in an automatic culture cell resulted in marked increases in the proportion of several mutants. The P1 phage-resistant cells increased 10 to 3000 times, the T2 phage-resistant cells 1 to 1000 times, the neomycin-resistant cells 1 to 10 times, and the virus-producing cells 30 to 70 times. No change occurred in the penicillin-resistant cells. Calculation of the growth curves and direct determination of the mutation rates by the null fraction method showed that the increases in the proportion of mutants were due to increases in the mutation rates.     

Evidence for the Direct Action of Colicin K on Aerobic 32Pi Uptake in Escherichia coli In Vivo and In Vitro

Pentachlorophenol (PCP)-sensitive incorporation of (32)P-labeled orthophosphate ((32)P(i)) into nucleotides and nucleic acids by disrupted spheroplasts of Escherichia coli was inhibited by addition of colicin K. Incorporation by intact cells was also inhibited by a similar concentration of colicin K. Various colicin K-resistant mutants were isolated, and their ability to incorporate (32)P(i) was tested. When T6(r)-colK(r) mutants (T6 phage-resistant) and tol I mutants (T6-sensitive, colicin E-sensitive) were converted to disrupted spheroplasts, their (32)P(i)-incorporation became sensitive to colicin K. On the contrary, incorporation by disrupted spheroplasts from tol II mutants (T6-sensitive, colicin E-resistant) was fairly resistant to colicin K like that of intact cells. A modification of the cell surface of T6(r)-colK(r) mutants, caused by mutation to novobiocin-permeable, T4 phage-resistant cells, restored the sensitivity of the cells to colicin K. The modified T6(r)-colK(r) cells did not adsorb T6 phage or colicin K, indicating that the receptors for T6 phage or colicin K are not reactivated by this modification. Similar treatment of tol I mutants did not have this effect. These observations strongly suggest that colicin K can act on its target on the cell membrane if it can penetrate the cell surface to reach this target. The receptor for colicin K on the cell surface, which may be part of the T6 phage-receptor, may have some unknown function in relation to the action of colicin K in normal cells, but tends to become dispensable if the cells become permeable to colicin K.     

寒地稻田不同灌溉模式下稻株生长与水分消耗利用

为揭示不同灌溉模式下水稻植株生长与水分消耗利用,通过蒸渗仪与田间小区结合的方法,以常规淹灌模式作为对比,研究了三种灌溉模式的水稻植株生长与水分消耗利用。试验结果表明：水稻植株体通过水分的自我调解来适应稻田生态系统变化,灌溉模式对水稻植株各器官的湿基含水率产生显著影响（P<0.05）,控制灌溉模式能够有效地延缓水稻生育后期的根系衰老;作为水分的载体,水稻植株干物质积累量直接影响水稻的耗水量,控制灌溉模式下稻田生态系统的水稻耗水量较间歇灌溉和常规淹灌都有大幅度的降低,而水分利用效率大幅度提高;控制灌溉模式可以通过生长补偿效应来增加后期干物质的积累,从而提高籽粒产量。根据各灌溉模式水稻的腾发量结合实际降雨量,来调控稻田灌溉水量,能够有效地维持SPAC稻田生态系统平衡,保证农业和生态系统的可持续发展。研究结果可为寒地黑土区稻田生态系统水分消耗利用研究提供理论依据和技术支撑。     

Immune islet killing mechanisms associated with insulin-dependent diabetes: in vitro expression of cellular and antibody-mediated islet cell cytotoxicity in humans

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Effect of Rural Sewage Irrigation Regime on Water-Nitrogen Utilization and Crop Growth of Paddy Rice in Southern China

Reclaimed water irrigation has become an effective mean to alleviate the contradiction between water availability and its consumption worldwide. In this study, three types of irrigation water sources (rural sewage’s primary treated water R1 and secondary treated water R2, and river water R3) meeting the requirements of water quality for farmland irrigation were selected, and three types of irrigation water levels (low water level W1 of 0–80 mm, medium water level W2 of 0–100 mm, and high water level W3 of 0–150 mm) were adopted to carry out research on the influence mechanismS of different irrigation water sources and water levels on water and nitrogen use and crop growth in paddy field. The water quantity indicators (irrigation times and irrigation volume), soil ammonium nitrogen (NH₄⁺-N) and nitrate nitrogen (NO₃⁻-N), rice yield indicators (thousand-grain weight, the number of grains per spike, and the number of effective spikes), and quality indicators (the amount of protein, amylose, vitamin C, nitrate and nitrite content) of rice were measured. The results showed that, the average irrigation volume under W3 was 2.4 and 1.9 times of that under W1 and W2, respectively. Compared with R3, the peak consumption of rice was lagged behind under R1 and R2, and the nitrogen form in 0–40 cm soil layers under rural sewage irrigation was mainly NH₄⁺-N. The changes of NO₃⁻-N and NH₄⁺-N in the 0–40 cm soil layer showed the trend of declining and then increasing. The water level control only had a significant effect on the change of NO₃⁻-N in the 60–80 cm soil layer. Both irrigation water use efficiency and crop water use efficiency were gradually reduced with the increase of field water level control. The nitrogen utilization efficiency under rural sewage irrigation was significantly higher than that under R3. Compared with the R3, rural sewage irrigation could significantly increase the yield of rice, and as the field water level rose, the effect of yield promotion was more obvious. It was noteworthy that the grain of rice under R1 monitored the low nitrate and nitrite content, but no nitrate and nitrite was discovered under R2 and R3. Therefore, reasonable rural sewage irrigation (R2) and medium water level (W2) were beneficial to improve nitrogen utilization efficiency, crop yield and crop quality promotion.

     

不同土壤水分处理对水稻光合特性及产量的影响

为探明土壤水分对水稻生长发育的影响机理,以武香粳14和两优培九为试验材料,分析了不同土壤水分处理下(W1、W2、W3和CK分别表示土壤体积含水量为20%、30%、40%和5cm水层灌溉)的水稻光合特性、产量及水分生产率等。结果表明,轻度水分胁迫(W3)具有处理间最大的叶片气孔导度、蒸腾速率和净光合速率,其他处理规律不显著。灌浆初期各水分处理下叶位间光合指标均表现为:剑叶＞顶2叶＞顶3叶>顶4叶,其他生育期规律不显著。与对照处理(CK)相比,武香粳14的W1、W2和W3处理的产量分别减少61.14%和29.13%、增加0.96%,水分生产率分别减少10.69%、增加1.53%和20.61%;两优培九的产量分别减少64.11%和28.76%,增加2.08%,水分生产率分别减少16.39%,增加2.46%和22.13%。研究结果为水稻精确灌溉和节水生产提供了技术支撑。     

Mechanisms of Resistance in Escherichia coli to the Sideromycin Antibiotic No. 216: Isolation and Characterization of the Resistant Mutants

Escherichia coli easily developed resistance to a new antimicrobial agent of the sideromycin group, No. 216, by spontaneous mutation. Most of the No. 216-resistant mutants tested proved not to be cross-resistant to E. coli phages T1, T5, and φ80. On the other hand, these phage-resistant mutants were cross resistant to No. 216. The initial site for binding of No. 216 to the sensitive cells was located, at the ton A gene product (Ton A-protein) of the outer membrane. However, unlike the phage-resistant mutants, ton A protein (78K-protein) in most No. 216-resistant mutants was intact and these mutants were possessed a particular 87K protein in the outer membrane. It is suggested that No. 216 is taken up by ton A protein and then penetrates into the cell by way of a particular transport system and that a highly mutable portion may exist in this reaction system.     

高产条件下不同小麦品种耗水特性和水分利用效率的差异

设置不灌水(W0)、底墒水+拔节水(W1)、底墒水+拔节水+开花水(W2)3个灌水处理,采用6个冬小麦(Triticum aestivum.L.)品种,研究了不同品种耗水特性和水分利用效率的差异.结果表明:(1)依据籽粒产量和水分利用效率2个因子,采用聚类分析的方法,将供试品种分为高水分利用效率组(Ⅰ组)、中水分利用效率组(Ⅱ组)和低水分利用效率组(Ⅲ组).同一灌水条件下的籽粒产量,Ⅰ组显著高于Ⅱ组和Ⅲ组;Ⅱ组和Ⅲ组在W0条件下无显著差异,在W1和W2条件下Ⅱ组显著高于Ⅲ组.(2)从Ⅰ组、Ⅱ组、Ⅲ组中分别取1个品种,泰山23、潍麦8号、山农12进一步分析表明,在W0 和W1条件下,泰山23和潍麦8号的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花,山农12为播种至拔节>开花至成熟>拔节至开花.W2条件下,3个品种的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花;播种至拔节和拔节至开花的耗水模系数为泰山23>山农12>潍麦8号,此阶段的耗水量和耗水强度为泰山23品种最高;开花至成熟的耗水模系数为潍麦8号>山农12 >泰山23,此阶段的耗水量和耗水强度为泰山23品种最低.(3) 在W0 和W1条件下,总耗水量和灌水量、降水量及土壤耗水量占总耗水量的百分率为泰山23品种居中;W2条件下,灌水量和降水量占总耗水量的百分率为泰山23>潍麦8号>山农12,土壤耗水量及其占总耗水量的百分率反之,但泰山23的总耗水量最低.(4) 同一灌水条件下,泰山23品种100～200cm土层的土壤耗水量高于潍麦8号,表明该品种能充分利用深层土壤水;山农12品种在W0和W2条件下,100～200 cm土层的土壤耗水量高于泰山23和潍麦8号,但其籽粒产量和水分利用效率显著低于上述两品种.     

Selection of phage-resistent clones of streptococcus group A and their properties]

In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.     

The growth characteristics and yield potential of rice (Oryza sativa) under non‐flooded irrigation in arid region

The objectives of this field experiment were to study the growth characteristics and yield potential of rice plants under non‐flooded irrigation in arid area. Non‐flooded treatments included drip irrigation with plastic mulching treatments (DIs), furrow irrigation with plastic mulching treatment (FIM) and furrow irrigation with non‐mulching treatment (FIN). Conventional flooded cultivation (F) was check treatment (CK). The four drip irrigation treatments differed in the amount of water applied before and after panicle initiation. Root length density, leaf dry weight, shoot dry weight and root activity were generally higher in the non‐flood‐irrigated treatments (especially the drip‐irrigated treatments) than in the flood‐irrigated treatment at mid‐tillering. However, the growth and development of rice plants were limited after jointing in the non‐flooded irrigation treatments. Increasing the root/shoot ratio and root length density in the 20–40 cm depth and decreasing specific root length at 0–20 cm soil layer were important mechanisms for helping the rice plants to adapt to the non‐flooded environmental stresses. Finally, the grain yield in the non‐flooded irrigation treatments was lower than that in the F treatment. These low yields were mainly attributed to the low root length density at 0–20 cm depth and root activity. Generally speaking, the restricted degrees in the DIs were smaller than that in the FIM and FIN treatments. Among the DIs, both the highest grain yield (8223–8900 kg ha^?1) and the highest water use efficiency (WUE) (0.63) were observed when the soil water content was kept at ?30 kPa before panicle initiation and at ?15 kPa after panicle initiation (referred to as the DI2 treatment). The yield in the DI2 treatment was not significantly different than that in the flood‐irrigated treatment. However, WUE was 2.5 times higher in the DI2 treatment than in the F treatment. These results suggest that drip irrigation technology can be considered as a better water‐saving cultivation of rice plants in arid region.     

Interaction of RNA-containing bacteriophages with host cell: MS2-induced mutants of <Emphasis Type="Italic">E. coli</Emphasis> and the occurrence of DNA-containing derivatives of the bacteriophage MS2

Sensitive cells of Escherichia coli AB 259 Hfr 3000 infected with RNA-containing phage MS2 produce phage particles and simultaneously continue to divide, thereby segregating sensitive cells capable of sustaining new cycles of infection. Multiplication of phage in sensitive cells gives rise to phage-resistant forms in the progeny of these cells. It is shown that this phenomenon is due not to selection of pre-existing phage-resistant mutants, but is instead the result of interaction between the phage and the cell. Unlike ordinary spontaneous or chemically induced E. coli mutants, MS2-induced phage-resistant cells are genetically unstable forms. In the course of reproduction they segregate new MS2-resistant forms with more highly expressed variations in the region encoded by the sex factors. Cells of the two final forms of MS2-induced mutants also produce a new type of phage. This new type constitutes DNA-containing forms which, however, are neutralized by anti-MS2-serum. The segregation of these forms serves to confirm that the genetic substance of the RNA-containing bacteriophage is capable of being expressed as a component of the DNA-containing structure.     

Comparison on physiological adaptation and phosphorus use efficiency of upland rice and lowland rice under alternate wetting and drying irrigation

As one of the most widely promoted water-saving irrigation strategies for rice, alternate wetting and drying irrigation (AWD) can not only save water but also increase mineral nutrient use efficiency. In this study, we compared the growth conditions of four rice varieties (two lowland and two upland varieties) under three irrigation regimes: continuously flooded (CF), alternate wetting and moderate soil drying (AWD15) and alternate wetting and severe soil drying (AWD30). AWD15 and AWD30 enabled the plants to receive fewer irrigation events and less irrigation water than CF, thereby saving both water resources and labor. AWD15 reduced redundant vegetative growth, promoted root growth, and increased the root-shoot ratio and harvest index. AWD15 increased the grain yield, water use efficiency (WUE) and phosphorus use efficiency (PUE) of upland rice and maintained the grain yield while increasing the WUE and PUE of lowland rice. More developed root systems under AWD helped upland rice to maintain a higher water status than lowland rice when plants were subjected to soil drying, which resulted in superior performance in grain yield in upland rice. AWD30 could not reconcile the demands of higher yield and the desire to reduce irrigation water use because it decreased grain yield. The results indicate that AWD15 irrigation of rice can not only increase rice yield and WUE but also enhance PUE, which can potentially reduce the use of phosphorus fertilizers. The results provide theoretical and technical support for improving rice cultivation.     

Type VI secretion systems of plant-pathogenic Burkholderia glumae BGR1 play a functionally distinct role in interspecies interactions and virulence

In the environment, bacteria show close association, such as interspecies interaction, with other bacteria as well as host organisms. The type VI secretion system (T6SS) in gram-negative bacteria is involved in bacterial competition or virulence. The plant pathogen Burkholderia glumae BGR1, causing bacterial panicle blight in rice, has four T6SS gene clusters. The presence of at least one T6SS gene cluster in an organism indicates its distinct role, like in the bacterial and eukaryotic cell targeting system. In this study, deletion mutants targeting four tssD genes, which encode the main component of T6SS needle formation, were constructed to functionally dissect the four T6SSs in B. glumae BGR1. We found that both T6SS group_4 and group_5, belonging to the eukaryotic targeting system, act independently as bacterial virulence factors toward host plants. In contrast, T6SS group_1 is involved in bacterial competition by exerting antibacterial effects. The ΔtssD1 mutant lost the antibacterial effect of T6SS group_1. The ΔtssD1 mutant showed similar virulence as the wild-type BGR1 in rice because the ΔtssD1 mutant, like the wild-type BGR1, still has key virulence factors such as toxin production towards rice. However, metagenomic analysis showed different bacterial communities in rice infected with the ΔtssD1 mutant compared to wild-type BGR1. In particular, the T6SS group_1 controls endophytic plant-associated bacteria such as Luteibacter and Dyella in rice plants and may have an advantage in competing with endophytic plant-associated bacteria for settlement inside rice plants in the environment. Thus, B. glumae BGR1 causes disease using T6SSs with functionally distinct roles.     

Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways

The vascular wilt fungus Fusarium oxysporum causes disease in a wide variety of crops. A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was previously found to be required for plant infection. To investigate the role of the heterotrimeric G-protein beta subunit Fgb1 as a putative upstream component of the Fmk1 signalling cascade, we generated F. oxysporum strains carrying either a Deltafgb1 loss-of-function allele or an fgb1(W115G) allele that mimicks the yeast STE4(W136G) mutation resulting in insensitivity to the cognate G-protein alpha subunit. Both types of mutants showed reduced virulence on tomato plants, similar to Deltafmk1 strains. However, in contrast to the latter, Deltafgb1 mutants displayed an abnormal hyphal growth phenotype with highly elongated cells, increased tip growth, a completely straight hyphal growth axis, and reduced subapical branching. Exogenous cAMP reversed part but not all of the Deltafgb1 growth phenotypes. Likewise, expression of the fgb1(W115G) allele only partly reversed growth phenotypes and failed to restore virulence on plants, whereas reintroduction of a functional fgb1 allele fully restored the wild type phenotype. Immunoblot analysis showed that levels of Fmk1 phosphorylation in fgb1 mutants were comparable to those in the wild type strain. Our results support a model in which Fgb1 controls hyphal growth, development and virulence in F. oxysporum both through cAMP-dependent and -independent pathways.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.