- Volume 138, Issue 3, 1992

Summary: Zoospores of the fungus Phytophthora palmivora undergoing synchronous differentiation showed a rapid increase (250%) in phosphatidic acid (PA) concentration within 20 s of the inducing stimulus. There were only small (< 100%) changes in cGMP and cAMP and inositol phosphates during the same period. There was no consistent change in the concentration of diacyl glycerols during the first five minutes of differentiation. The addition of exogenous PA (3 mUm) induced zoospore differentiation, with the optimum concentration dependent upon the Ca2+ concentration in the suspension medium. Other phospholipids were ineffective as inducers. Both PA production and spore differentiation were Ca2+-dependent, and the addition of the Ca2+ channel blocker verapamil, or the removal of exogenous Ca2+ by EGTA addition, both reduced PA accumulation and slowed differentiation. We suggest that PA production in this organism arises via a stimulus-activated phospholipase D, and may act as a second messenger. There is no evidence for any role for cyclic nucleotides or inositol phosphates as second messengers during the early events of zoospore differentiation in this species.

Summary: Copper-zinc superoxide dismutases ([Cu,Zn]-SODs) are ubiquitous in eukaryotes but have rarely been found in prokaryotes. A gene for [Cu,Zn]-SOD (sodC) has recently been cloned from Haemophilus influenzae type b and H. parainfluenzae, so other Haemophilus and related species were screened for the presence of [Cu,Zn[-SODs by visualization of bands of SOD activity in non-denaturing polyacrylamide gels and by gene probing. Strains of H. aphrophilus, H. paraphrophilus, H. haemolyticus, H. paraphrohaemolyticus, some non-typable H. influenzae, H. haemoglobinophilus (canis) and H. parasuis were all found to have [Cu,Zn]-SOD activity (inhibited by 2 mm-cyanide) in polyacrylamide gels. In a Southern blot analysis, DNA from H. aphrophilus, H. paraphrophilus, H. haemolyticus and [Cu,Zn]-SOD-containing non-typable H. influenzae - but not the other species - hybridized to a 360 nucleotide DNA probe containing the 5′-part of sodC cloned from H. influenzae type b. Bacterial [Cu,Zn]-SODs are more prevalent than has previously been recognized.

Summary: The soluble haemagglutinins produced by plasmodia of Physarum polycephalum were purified by chromatographic methods and resolved into haemagglutinins I and II. On SDS-PAGE, purified haemagglutinins I and II each gave a single band with an apparent molecular mass of 6 and 11 kDa, respectively. The results of gel-filtration chromatography suggested that both haemagglutinins were dimers of the respective subunits under non-denaturing conditions. Rabbit erythrocytes were preferentially agglutinated by both haemagglutinins. The human type A, B and O erythrocytes were agglutinated by haemagglutinin II to an equal degree but were not agglutinated by haemagglutinin I. Simple sugars failed to inhibit the activities of both haemagglutinins. The activities, however, were effectively inhibited by the addition of thyroglobulin. Other glycoproteins such as fetuin, orsomucoid and transferrin inhibited the activity of haemagglutinin I but not that of haemagglutinin II. These haemagglutinins were detected in a slime fraction obtained from the culture media of starved plasmodia, suggesting that they are released to the outside of the plasmalemma to become associated with the slime layer on the plasmodial surface.

Summary: In a previous study, electron microscopic examinations of thin sections of Lactobacillus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identified as a layer of a non-glycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (S-layer) lattice (a = 4·5 nm, b = 9·6 nm, γ = 77 °) which completely covers the cell surface. Treatment with 5 m-LiCl extracted the S-layer protein from intact cells efficiently and selectively. Viability did not decrease significantly. Moreover, the S-layer reappeared when treated cells were allowed to grow again. In vitro self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in vivo. The purified S-layer protein had a high content (44%) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid.

Summary: The pelA gene from Erwinia chrysanthemi strain 3937, which encodes the acidic pectate lyase, PLa, has been sequenced and characterized. The structural gene consists of a 1179 bp open reading frame encoding a polypeptide of 41555 Da, which includes an N-terminal signal peptide. The deduced amino acid sequence shows a protein very similar to some PLs already sequenced. Cloning of the pelA gene behind the lacZ promoter of the vector pTZ19R allowed overexpression of PLa into a derivative of strain 3937 deleted of the other pel genes. The mature protein was obtained in milligram amounts from the supernatant of this strain and at homogeneous purity after two purification steps. Its biochemical properties were similar to those of other PLs. Polyclonal antibodies raised against the purified PLa cross-reacted with the basic pectate lyase PLd, but not with PLe. The role of PLa in pathogenicity is discussed.

Summary: Transposon Tn916 was shown to be capable of direct conjugative transfer in broth and membrane matings between strains of Escherichia coli K12 and between E. coli K12 and Haemophilus influenzae type b. Only Tn916 was transferred, but Tn916 donor ability was not itself inheritable by the recipients and seemed to be associated with the presence of Tn916 on a non-conjugative pBR322-derived vector in the original donor strain. Transfer of Tn916 by conjugation was found to be an efficient method for producing insertion mutations in the chromosome of recipient cells. Although such insertions were unstable when the cells were grown under non-selective conditions, it was possible to show that over 40% of the isolated Tn916 insertions in the chromosome of E. coli K12 were in gene(s) concerned with histidine biosynthesis, implying that there is a partial hot-spot for Tn916 insertion on the E. coli K12 chromosome. When a strain of H. influenzae type b was used as a recipient, out of approximately 1500 transconjugants tested, two mutants were isolated with insertions in genes controlling the expression of iron-regulated transferrin-binding proteins. These mutants constitutively produced major 76 kDa and minor 90 kDa proteins which bound transferrin, even when grown under iron-sufficient conditions. Tn916 insertion mutagenesis, following transfer by conjugation, is a convenient method for isolating mutations in genes concerned with iron acquisition by this important human pathogen.

Summary: A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 °). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 °), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.

Summary: Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.

Summary: The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa. Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity. The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis. We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin. Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease. Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection. The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents.

Summary: Heterotrophic pyruvate-limited steady-state continuous cultures of the bacterium Aquaspirillum autotrophicum were perturbed with a pulse injection of a small volume of concentrated pyruvate solution. These cultures exhibited an instantaneous change in the growth dynamics, turning from steady state to apparently linear growth. These transient growth-responses had no lag phase and were clearly distinct from unlimited exponential growth according to the initial rates of increase of biomass and substrate disappearance kinetics. A linear accumulation with time of poly(β-hydroxybutyrate) was observed within the cells. Slopes of these linear responses were negatively correlated with the dilution rate. Physiological bases of linear growth are discussed in the light of the models of H. E. Kubitschek. Poly(β-hydroxybutyrate) synthesis in the absence of exogenous limitation may serve to protect the cells against a transient metabolic overflow.

Summary: The interchange of octadecenoic acids and dihydrosterulic acid was a response of aerobically growing Lactobacillus fermentum to changes in growth temperature. Oleic and vaccenic acid contents decreased both at temperatures below 20 ° and above 26 °, showing mirror image behaviour, with a concomitant increase in dihydrosterulic acid. A temperature-dependent shift from vaccenic to oleic acid synthesis, and the conversion of the latter to dihydrosterulic acid was responsible for the overall change. Consequently, the degree of fatty acid unsaturation decreased at temperatures above 26 °, whereas the degree of cyclization increased. The converse occurred below 20 °. The relative amount of lactobacillic acid, total cellular fatty acid content, and mean fatty acid chain length were practically temperature-independent. The occurrence of oleic acid is thought to be related to aerobic growth conditions.

Summary: Photosynthetic activities of a synchronously grown aerobic N2-fixing unicellular cyanobacterium, Synechococcus sp. strain Miami BG 043511, were examined. Cells 1 h after the onset of synchronous growth exhibited an O2 evolution rate of 400-500 mUmol (mg Chl aL)-1 h-1 at saturating light intensity, while it decreased to 40-80 mUmol (mg Chl a)-1 h-1 in 12 h cells. Changes in the relative fluorescence intensity (excitation wavelength 430 nm, emission 685 nm) were not as large as the changes in photosynthetic O2 evolution activity. Photosynthetic electron transport activities of both photosystem II and photosystem I in 12 h cells were lower than those in 1 h cells. Photosystem II activity with 2,5-dimethyl-p-benzoquinone, and photosystem I activity with diaminodurene and methyl viologen in 12 h cells were about 64 and 46%, respectively, of those in 1 h cells. However, the reduction in photosynthetic electron transport activities was not sufficient to account for the reduction in photosynthetic O2 evolution activity in 12 h cells, which was about 14% of that in 1 h cells. Based on these observations, the reduction in photosynthetic O2 evolution activity in 12 h cells was ascribed to the combined effect of a reduction in both the photochemical and the biochemical steps of photosynthesis.

Summary: The regulation of nitrogenase derepression, plus the catalytic activity and protein concentration of glutamine synthetase (GS), nitrate reductase (NR), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phycoerythrin (PE) were studied in the filamentous non-heterocystous cyanobacterium Plectonema boryanum PCC 73110. Both nitrogen limitation and microaerobic incubation were essential for the derepression of nitrogenase. Oxygen caused irreversible inactivation of nitrogenase, as well as repression of its synthesis. A temporal separation of N2 fixation and net photosynthetic O2 evolution was observed under a N2/CO2 (95:5, v/v) atmosphere. Repeated peaks of nitrogenase and growth were observed. Immunogold localization showed that in N2-fixing cultures, all cells, including those undergoing division, contained nitrogenase, and that the nitrogenase antigen was uniformly distributed throughout the cells without any preferential association with cellular structures. Rubisco was mainly located in carboxysomes of both N2-fixing and NO- 3-grown cells. Both N2-fixing and NO- 3 -grown cells showed similar levels of PE, which was associated with the thylakoid membranes. GS antigen was distributed throughout the cells and the relative amounts of this enzyme, as well as its activity, were 20% higher in N2-fixing than in NO- 3-grown cultures. NO- 3 uptake and NR systems were found to be NO- 3 inducible, with very low activities in N2-fixing cultures. The latter may be important in avoiding competition for Mo between nitrogenase and NR.