The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.     

Reproductive pattern,perinatal mortality,and sex preference in rural Tamil Nadu,south India: community based,cross sectional study.

OBJECTIVES: To study reproductive pattern and perinatal mortality in rural Tamil Nadu, South India. DESIGN: Community based, cross sectional questionnaire study of 30 randomly selected areas served by health subcentres. SETTING: Rural parts of Salem District, Tamil Nadu, South India. SUBJECTS: 1321 women and their offspring delivered in the 6 months before the interview. MAIN OUTCOME MEASURES: Number of pregnancies, pregnancy outcome, spacing of pregnancies, sex of offspring, perinatal and neonatal mortality rates. RESULTS: 41% of the women (535) were primiparous; 7 women (0.5%) were grand multiparous (> 6 births). The women had a mean age of 22 years and a mean of 2.3 pregnancies and 1.8 live children. The sex ratio at birth of the index children was 107 boys per 100 girls. The stillbirth rate was 13.5/1000 births, the neonatal mortality rate was 35.3/1000, and the perinatal mortality rate was 42.0/1000. Girls had an excess neonatal mortality (rate ratio 3.42%; 95% confidence interval 1.68 to 6.98; this was most pronounced among girls born to multiparous women with no living sons (rate ratio 15.48 (2.04 to 177.73) v 1.87 (0.63 to 5.58) in multiparous women with at least one son alive). CONCLUSIONS: In this rural part of Tamil Nadu, women had a controlled reproductive pattern. The excess neonatal mortality among girls constitutes about one third of the perinatal mortality rate. It seems to be linked to a preference for sons and should therefore be addressed through a holistic societal approach rather than through specific healthcare measures.     

Effect of vegetation on density thresholds of adult desert locust gregarization from survey data in Mauritania

In the desert locust, Schistocerca gregaria (Forskål) (Orthoptera: Acrididae), the threshold density inducing the gregarization phenomenon has never been determined under natural conditions. The influence of environmental factors on this phenomenon has been studied mostly in controlled environments. Based on data collected during several years by the survey teams of the National Center for Locust Control in Mauritania, we analyzed the influence of locust density, vegetation cover, and vegetation status on the probability of observing gregarious locusts. We assumed that a probability to observe gregarious locusts of 0.5 corresponded to the density threshold of gregarization. The results showed in detail the change in the threshold of gregarization according to the cover and status of the vegetation. Low cover and dry vegetation led to a low density threshold of gregarization probably due to high probability of individuals to touch each other. Dense and green vegetation favored a high threshold of gregarization probably due to a dispersion of the individuals and a low probability of individual encounters. These findings should help the management of locusts and decision making during control operations.     

Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis.

A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.