Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora.

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.

Glycan engineering of proteins with whole living yeast cells expressing rat liver alpha2,3-sialytransferase in the porous cell wall.

The N-glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases. Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals. We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver alpha2,3-sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins. The Km values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N. The yeast strain provides an inexpensive self-perpetuating source of ST3N activity for glycan engineering of recombinant proteins.

Vitamin D treatment and bone mineral density in the aged.

The purpose of this study was to investigate whether long-term vitamin D treatment increased bone mineral density in the aged. The bone mineral density in the distal forearm and femoral neck did not differ between nine residents (mean age 81.0 years) of an old peoples' home who had received an annual injection of 150,000 IU ergocalciferol during the foregoing 2-7 years (mean 5.1 years) and nine age-, weight- and height-matched control subjects who had subnormal 25 hydroxyvitamin D level. The alkaline phosphatase and parathyroid hormone levels were clearly higher when the 25 hydroxyvitamin D level was below 10 nmol/1. The authors suggest that the ability of vitamin D treatment to diminish fracture incidence may derive from improved bone quality, not measurable by the standard dual energy X-ray absorptiometry, and/or improved nervous and muscular control of movements to counter the tendency to fall.

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity.

The celV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 bp and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of CelV1 was highly similar to that of CelV of another E. c. subsp. carotovora strain SCR1193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. CelV1 mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora.

The pectate lyase (Pel, EC 4.2.2.2) isoenzyme profile of Erwinia carotovora subsp. carotovora was characterized by isoelectric focusing, and the corresponding genes coding for four different exported Pels were cloned. The nucleotide sequence of the pelB gene encoding one of these isoenzymes was determined and was shown to contain 1,040-bp open reading frame coding for a 37,482-Da protein with a putative cleavable amino terminal signal peptide. Overexpression and selective labeling experiments with the pelB clone demonstrated the synthesis of a 35-kDa polypeptide, which is in accordance with the deduced size of the processed PelB. The predicted amino acid sequence of PelB was very similar to that of Pel-3 of another E.c. subsp. carotovora strain 71, but showed no similarity to other previously characterized pectinolytic enzymes. The pelB gene is located next to the previously characterized pehA gene encoding an endopolygalacturonase. The two genes are divergently transcribed from a common control region and are subject to similar global regulation by the central virulence regulator expI. Inactivation of pelB did not appear to reduce the virulence of the mutant strain, suggesting that pelB does not have a major role in pathogenicity. Unlike other Pels, PelB required partially methyl esterified pectin as substrate suggesting that PelB represents a novel isoform of pectate lyase.

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.

Annual injection of vitamin D and fractures of aged bones.

In order to investigate the effect of a supplementation of vitamin D in the prophylaxis of fractures of the bones of aged people, an annual intramuscular injection of ergocalciferol (150,000-300,000 IU) was given to two series of aged subjects: first to 199 (45 male) of 479 subjects (110 male) aged more than 85 years who were living in their own home, and second to 142 (29 male) of 320 (58 male) subjects aged 75-84 and living in a home for aged people. This prospective series was divided into treatment groups according to month of birth. These injections were given annually from September to December in the years 1985-1989, two to five times to each participant. The fracture rates, laboratory values, vitamin D levels, possible side effects, and mortality were followed until October 1990. A total of 56 fractures occurred in the 341 vitamin D recipients (16.4%) and 100 in 458 controls (21.8%) (P = 0.034). The fracture rate was about the same in both outpatient and municipal home series. Fractures of the upper limb were fewer in the vitamin D recipients, 10/341 = 2.9% (P = 0.025), than in the controls, 28/458 = 6.1%, during the follow-up. A similar result was obtained in fractures of ribs, 3/341 = 0.9% and 12/458 = 2.6%, respectively. Fractures of the lower limbs occurred almost as frequently, 31/341 = 9.1%, among the vitamin D recipients as among the controls, 49/458 = 10.7%. The fracture rate was higher in females (22.2%) than in males (9.5%). The fractures were fewer in the vitamin D recipients only in females.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein.

The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp. carotovora has been cloned previously [Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044]. We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens. To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions. Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence. There was approximately fourfold variation in the production efficiency of B. subtilis strains carrying the different constructs. The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide. This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium. In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs. Our results suggest that fusions containing intact signal peptides, which are common when producing foreign proteins, are not necessarily the most efficient.

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis.

The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-poly-acrylamide gel electrophoresis characterizations. The NH2-terminal sequence analysis of the secreted PME revealed two different NH2-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and alpha-amylase signal peptide.

Serum vitamin D level after an annual intramuscular injection of ergocalciferol.

An annual intramuscular injection of ergocalciferol (150,000 IU) normalized low serum (25(OH)D concentrations in elderly people for 1 year. The treatment had a slight effect on serum 24,25(OH)2D levels but no effect on 1,25(OH)2D levels.

Body weight and the three-year prognosis in very old people.

We studied prospectively the three-year prognosis of persons aged 84-88 years living in their own homes in the city of Tampere, Finland in 1981-84. Out of the target population 722 persons (64 per cent), 181 of them male (25 per cent), participated in the initial survey. A re-examination was carried out annually for the next three years. Increased mortality was observed for subjects with body mass index less than or equal to 22.0 kg/m2, statistically significantly in women but not in men. Mortality was not increased in those with body mass index greater than or equal to 30.0 kg/m2 in either sex. Low body mass index was associated with mortality due to stroke. Low body mass index was related to permanent hospitalization and also to failure to cope with living at home. On average, body weight was somewhat reduced during the follow-up period. Both a loss and a gain in weight by two kilograms or more during the first follow-up year were associated with increased mortality but not with institutionalization during the next two years.

Blood pressure in the very old.

Seven hundred and twenty-four people (541 female, 183 male), aged 84-88 years and living at home in the city of Tampere, Finland, accepted our invitation to be examined in the outpatient department of the local geriatric hospital. These subjects were re-examined annually, and their fate was followed for 3 years. The blood pressure level was a prognostic sign for mortality during the first year after its measurement. The lowest mortality rate was found among those subjects with systolic blood pressure between 140 and 169 mmHg and diastolic blood pressure between 70 and 99 mmHg. Blood pressures outside of these ranges were associated with increased mortality rates, particularly cardiac and coronary mortality. The mortality rate was especially high among those whose systolic blood pressure had decreased to low levels in the year before, but was lower in those whose systolic blood pressure had decreased from high to middle levels (140-169 mmHg) than in those with consistently high systolic blood pressure. Eventual institutionalization was more common in those with low systolic blood pressure.

Blood pressure and five year survival in the very old.

During 1977-8 we measured blood pressure in 561 old people (83% of those aged 85 or more living in Tampere) and analysed their five year survival according to their blood pressure group. The subjects were divided into six groups on the basis of their blood pressures (from less than 120 to greater than 200 mm Hg systolic, from less than 70 to greater than 110 diastolic). The greatest mortality was observed in those in the lowest systolic and lowest diastolic groups. Mortality was least in subjects with systolic pressures of 160 mm Hg or more and diastolic pressures of 90 mm Hg or more. The most essential finding in this series of the very old was an increased mortality in the lowest blood pressure groups.

Screening of thyroid function in the hospitalized aged.

When a thyroid test was made on all the patients coming to a geriatric department of a hospital, markedly more new cases of hypothyroidism (17 as against 2), and also somewhat more new cases of hyperthyroidism (34 as against 21) were found than when the thyroid test was used only on clinical suspicion of thyroid dysfunction. The authors consider a thyroid test (T4?) worth taking systematically in all aged patients admitted to hospital.

Blood glucose and diabetes mellitus predicting mortality in persons aged 85 years or above.

The population aged 85 years or over (n = 674) living in Tampere, Finland was surveyed in 1977-1978. In an investigation of the prognostic survival of new and previously diagnosed diabetic patients, the levels of blood glucose were analysed in 558 persons, 99 men and 459 women. The relative sex- and age-adjusted survival rates were evaluated at the 5-year follow-up. The mortality after 5 years of the 17 new diabetics at home did not differ significantly from that of the 225 non-diabetics at home. The mortality of 30 patients with previously diagnosed diabetes mellitus was higher than that of the non-diabetics. The survival prognosis of the diabetics on antidiabetic medication did not differ from that of those on diet. An increased risk of mortality was found in this series in previously diagnosed diabetics and--most unexpectedly--in non-diabetics with the lowest fasting blood-glucose levels.

Living conditions and health of a population aged 85 years or over: a five-year follow-up study.

The population aged 85 years or over (n = 674) living in Tampere, Finland was surveyed in 1977-78. Altogether 561 persons (83%), 99 men and 462 women, were examined. A re-examination of 170 persons, 23 men and 147 women, was carried out in 1982 by the same procedure as in 1977-78, The results were compared with those of the same people and with those of over-90s five years before. Of the group followed, in the initial survey 65% were living at home and 25% in old people's homes, 10% being hospitalized. The percentage of hospital in-patients had increased threefold during the five-year follow-up. The functional capacity of the subjects had deteriorated as regards mental function and ability to walk. The mean values of blood samples (apart from ESR) were still in normal ranges, although the levels of haemoglobin and haematocrit had fallen significantly. Dementia or confusion, anaemia, femoral-neck fracture and cataract were significantly increased.

Mortality and high haematrocrit in the very aged.

A series of 543 people, aged 85 years or more was studied and divided into groups according to haematocrit value. The mortality of the subjects was followed over the next 5 years. The mortality in the lowest haematocrit groups was highest during the whole observation period. Further, in the haematocrit bracket greater than or equal to 0.48, the mortality in the first year was higher than in those with haematocrit 0.40-0.47. In the second year this increased mortality had disappeared and in the third-fifth years decreased mortality was found with high haematocrit. Initially high haematocrit also decreased during the observation period. Since the increased mortality associated with high haematocrit disappeared it cannot be based on any chronic disease but on some disappearing factor, e.g. simply the high haematocrit itself.