Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes.

BACKGROUND AND AIMS: Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. METHODS: Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. KEY RESULTS: Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. CONCLUSIONS: Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.

Legume root nodule bacteria and acid pH.

In 1984 the Australian Wool Research Trust Fund called for expressions of interest in projects directed at using the developing techniques of molecular biology for application to agricultural problems. With our interests in legume root nodule bacteria and their physiology, we felt that the problems for legume nodulation and N2 fixation posed by soils which were already acid, or which were rapidly acidifying, required just such attention. Further, the finding body's request coincided with the highly successful introduction into Western Australian agriculture of acid-tolerant strains of the medic-nodulating bacteria Sinorhizobium meliloti originating from acid soils on Sardinia (see below). The existence of such strains made it obvious that acid tolerance was a genetically determined trait, and provided invaluable biologically diverse material with which to work. The biological bases for that trait of acid tolerance were totally obscure, and many remain so, but the following account provides some light in the darkness. The research that we have done since in pursuit of explanations for acid tolerance have been funded first by the Wool Research Trust Fund and the Rural Credits Development Fund, and later by the Australian Research Council, and we here record our appreciation for their support.

Acid tolerance in root nodule bacteria.

Biological nitrogen fixation, especially via the legume Rhizobium symbiosis, is important for world agriculture. The productivity of legume crops and pastures is significantly affected by soil acidity; in some cases it is the prokaryotic partner that is pH sensitive. Growth of Rhizobium is adversely affected by low pH, especially in the 'acid stress zone'. Rhizobia exhibit an adaptive acid tolerance response (ATR) that is influenced by calcium concentration. Using Tn5-mutagenesis, gusA fusions and 'proteome' analysis, we have identified a range of genes that are essential for growth at low pH (such as actA, actP, exoR, actR and actS). At least three regulatory systems exist. The two-component sensor-regulator system, actSR, is essential for induction of the adaptive ATR. Two other regulatory circuits exist that are independent of ActR. One system involves the low pH-induced regulator gene, phrR, which may control other low pH-regulated genes. The other circuit, involving a regulator that is yet unidentified, controls the expression of a pH-regulated structural gene (lpiA). We have used pH-responsive gusA fusions to identify acid-inducible genes (such as lpiA), and then attempted to identify the regulators of these genes. The emerging picture is of a relatively complex set of systems that respond to external pH.

A helicase gene (helO) in Rhizobium meliloti WSM419.

A 2.8 kb BamHI DNA fragment adjacent to a BamHI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced. A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with 'DEAH' motif helicases. Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene. Inactivation of helO by insertion of a omega interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc.

Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system.

An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (designated actS) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into actS in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (ActS-) and TG5-46 (ActR-), while fragments containing only actR or actS complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complemented not only TG5-46 but also RT295. Cloning DNA upstream from actR and actS into a broad-host-range lacZ expression vector and measuring beta-galactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria.

Respiratory function after cardiopulmonary bypass: a comparison of bubble and membrane oxygenators.

A consecutive sample of 500 adults undergoing cardiac surgery was randomly allocated to extracorporeal circulation with either a Bard bubble oxygenator H1700 or a Bard membrane oxygenator HF5700 (Bard Ltd, Crawley, UK). Alveolar-arterial oxygen tension gradient (AaDO2) was calculated prebypass, then 20, 90, 180, and 420 minutes postbypass. Preoperative, initial postoperative, and first-day postoperative chest x-rays were assigned an extravascular lung water (EVLW) score and an atelectasis score. There was a comparable increase in AaDO2 after bypass in each group. The increase in EVLW score was significantly greater in the bubble group (mean 2.91, 95% CI 2.28-3.54) than the membrane group (mean 2.06, 95% CI 1.43-2.69) for the initial postoperative x-rays (P < 0.01) and also for the x-rays on the first postoperative day (P < 0.01). The increase in atelectasis score was significantly greater in the bubble group (mean 1.06, 95% CI 0.94-1.18) than the membrane group (mean 0.86, 95% CI 0.74-0.98) for the initial postoperative x-rays (P < 0.01) but not for the x-rays on the first postoperative day. There was no difference in duration of ventilation, intensive care, hospital stay, or hospital mortality between bubble and membrane groups. Although there was a statistically significant difference in x-ray scores between oxygenator groups, neither intrapulmonary shunting nor clinical outcome was influenced by the type of oxygenator used during bypass.

Effects of adding an Italian theme to a restaurant on the perceived ethnicity, acceptability, and selection of foods.

We investigated whether a change in the perceived ethnicity of a food can be produced without manipulating the food item itself, and if that change in ethnic perception is accompanied by a change in acceptability and food selection behavior. Italian and British foods were offered in a British restaurant for four days. Foods were offered for 2 days under control conditions, when the restaurant was decorated as usual. The identical foods then were offered in the restaurant for 2 more days under experimental conditions, when ethnic names were used on the menu to describe foods, and the restaurant was decorated with an Italian theme. Perceived ethnicity and acceptability of items were rated by customers each day, and item selection was tracked. The Italian theme increased selection of pasta and dessert items, and decreased the selection of fish. The Italian theme also increased the perceived Italian ethnicity of British pasta items, fish and veal, and increased the perceived Italian ethnicity of the meal overall. These findings show that changes in perceived ethnicity and food selection can be accomplished without altering food items, but merely by manipulating the environment, and may imply a unique strategy for increasing perceived menu variety.

Intraventricular diamorphine via an Ommaya shunt for intractable cancer pain.

We describe two patients in whom diamorphine was administered into the intraventricular space via an Ommaya reservoir, producing excellent pain relief. The use of this technique for long term administration of analgesia is reviewed.

Blood pressure measurement during transport. A comparison of direct and oscillotonometric readings in critically ill patients.

This study assessed the accuracy of oscillotonometric blood pressure measurement in critically ill patients during transfer. Direct intra-arterial pressure measurements were taken in 44 transported patients as a 'gold standard' and compared with readings from four portable automatic oscillotonometers--the Dinamap 8100, Lifestat 100, Propaq 102 and Takeda UA711. All under-read systolic pressure (by 13%, 21%, 19% and 13% respectively) and over-read diastolic pressure (by 15%, 5%, 27% and 15% respectively) in comparison to direct pressure measurement. The limits of agreement for 95% of comparisons were broad. The systematic difference between direct and oscillotonometric measurements and the variability between and within patients show that direct oscillotonometric measurements are not interchangeable in these patients. Portable automatic oscillotonometers should not be substituted for direct monitoring in such circumstances.

Current practice in transferring critically ill patients among hospitals in the west of Scotland.

OBJECTIVE: To identify the requirements of an interhospital transfer service for critically ill patients. DESIGN: Retrospective survey of the current functions of a specialist interhospital transfer team from data collected at the time of transfer and from records of intensive care unit. SETTING: Mobile intensive care unit based at a tertiary referral centre, which serves the west of Scotland. PATIENTS: All critically ill patients (378) transferred between hospitals by the unit from 1986 to 1988. RESULTS: 365 Patients were transferred by road and 13 by air. There was a wide variation in age (range 6 weeks to 87 years), diagnosis, reason for transfer, support required, and distance travelled. Most patients (232) were transferred for respiratory or cardiovascular support; 100 were trauma cases. 300 Patients (79%) were mechanically ventilated during transfer. No patient died in transit, although the eventual mortality was 28% (105 patients). Mortality was significantly higher in patients transferred from hospitals with intensive care units than from those without (38% (125 patients) v 23% (253); p less than 0.005). IMPLICATIONS: Safe interhospital transfer of critically ill patients is feasible; the high eventual mortality in some patient groups emphasises the need for accurate prediction of outcome if inappropriate transfer is to be avoided. The findings may help in organising secondary transfer services in future.