- Volume 130, Issue 6, 1984

We studied the adherence of Haemophilus influenzae to monkey respiratory mucosa using nasal turbinates maintained in organ culture. Adherence of capsulated and rough strains was not inhibited by monosaccharides, sucrose, human albumin, foetal calf serum or polyribophos-phate. However, antisera directed against surface components decreased bacterial adherence. Although variation in adherence capacity in individual strains was observed there was no correlation with capsulation, anatomical site of strain isolation or biotype. Bacterial surface structures other than capsular material appear important in effecting upper respiratory tract colonization.

An immunogold labelling technique was used to label the pili of the bacterium Bacteroides nodosus. The labelling was distinct and highly specific, and individual pili could be recognised beneath the gold probe. The labelling of somatic antigens could be distinguished from that of pilus antigens. Furthermore, labelling of fragments of cytoplasm released by cell lysis and trapped in the pili could be distinguished from pilus labelling. An antiserum that had been raised against strain 80200 (serotype N) labelled pili of strain 215 (serotype B). Double labelling experiments with this antiserum and the antiserum against strain 215 (serotype B) showed that both antisera label the same pili bundles. The ease of detection of the immunocytochemical reaction should enable this technique to be used as a routine screen for pilus antigens. It also possesses the potential for much wider applications for immunolabelling other antigens, such as viruses, that can be obtained in suspension.

The effects of starvation on the arginine transport system in ANT-300, a psychrophilic marine Vibrio sp., were investigated. A rapid rate of uptake was detected on the first day of starvation, followed by a much lower but constant rate of uptake until day 35. Transport was found to require maintenance of a proton gradient across the membrane, but not to involve ATP. Respiration of the transported arginine increased during the 35 d of starvation. Thus, ANT-300 is able to maintain a high-affinity, active transport system for arginine during extended periods of starvation even in the absence of an exogenously supplied energy source.

Mixotrophic growth of a thermoacidophilic iron-oxidizing, facultatively autotrophic bacterium is described. Rapid growth with sucrose, fructose, glucose and ribose required the concurrent oxidation of ferrous iron, which, it was concluded, provided most of the energy required for biosynthesis. Specific iron oxidation rate during growth and biomass production depended on the sugar supplied. About 20% of the cell carbon was obtained from CO2-fixation during growth on glucose. The presence of ribulosebisphosphate carboxylase indicated this to be fixed by the Calvin cycle. The kinetics and inhibition of glucose transport indicated uptake by diffusion and by energy-dependent processes. Sensitivity to fluoride indicated the possible involvement of a phosphoenolpyruvate-phosphotransferase system. Radiorespirometry, using specifically labelled glucose molecules, demonstrated that glucose was oxidized by the oxidative pentose phosphate cycle with further oxidation of glyceraldehyde 3-phosphate by means of the tricarboxylic acid cycle. Dehydrogenases for glucose 6-phosphate and 6-phosphogluconate were present but 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase were not detected in cell-free extracts, showing absence of the Entner-Doudoroff pathway.

The role of phospholipid metabolism in the functioning of the bacterial envelope was investigated in the chain-forming Escherichia coli envC. Lysophosphatidylethanolamine (LPE) which accumulated in this strain during growth was indentified as the product of phosphatidylethanolamine (PE) hydrolysis by a phospholipase A1, i.e. 2-acylLPE. Isotopically labelled LPE transferred into intact mutant and parent cells by liposome/bacteria interaction was rapidly reacylated to PE. However, in envC the final PE/LPE ratio was lower than that in the parent, thus showing that the fate of LPE is modified. Crude cell extracts degraded LPE to a lesser extent in envC than in the parent but were unable to promote reacylation activity under our experimental conditions. In both strains, the lysophospholipase activity was neither calcium-dependent nor inhibited by the SH-group inhibitors p HMB or p CMPS, and hydrolysed 1-acylLPE as well as 2-acylLPE. These results indicate the existence of a deacylation-reacylation cycle in E. coli and show that this cycle is perturbed in envC cells, especially at the lysophospholipase step.

Acanthamoeba castellanii (CCAP 1534/3) was found to bind avidly the common soil bacterium Pseudomonas fluorescens. This adhesion was mediated not by pili nor by the general bacterial surface but by the polar flagella. Because of the nature of the flagellar rotary motor, the cell bodies of the attached bacteria could be seen rotating clearly. While initially bacterial binding occurred uniformly over the cell membrane of Acanthamoeba. the bacteria were soon swept posteriorly to form a cap and either endocytosed or sloughed off, still agglutinated by their flagella. Such capped amoebae would not bind Pseudomonas if challenged immediately, indicating a depletion of flagella-binding sites. The bacteria could not bind to amoebae pre-treated with concanavalin A (Con A) even after the lectin had been capped to the uroid. However, capping of flagella-binding sites did not co-cap all the Con A-binding sites on the surface of the amoeba. The flagella-binding sites were not affected by pre-treatment with Pronase (1 mg ml−1) or anti-Acanthamoeba surface antibody. Proteus mirabilis also bound avidly by its flagella to Acanthamoeba and, furthermore, competition experiments suggested that Proteus and Pseudomonas adhere to a common surface site on the amoeba. The presence of sites on the cell membrane of A. castellanii that are specific for flagellin would enhance strongly the adsorption of motile bacteria prior to endocytosis. This would represent an excellent feeding strategy for a soil-dwelling phagotroph.

Further evidence that kappa endosymbionts of stock 7 Paramecium biaurelia[Caedibacter varicaedens] are bacteria is given by the discovery of a prokaryotic translational system in these particles. Kappa proteins incorporate 35S in in vivo experiments. However, there is no evidence that the translational systems of the host paramecia and the symbionts are shared. On the contrary, experiments with symbiont-bearing and symbiont-free strains strongly indicate that all major proteins found in kappa are synthesized in kappa. In this respect, kappa clearly differs from cell organelles. Protein synthesis in kappa is not inhibited by cycloheximide and the aminoglycoside G418, which do inhibit, although not completely, protein synthesis of the host cells. On the other hand, chloramphenicol quantitatively blocks translation in kappa without any obvious effects on the translational system of the paramecium cytoplasm.

Stock 7 kappa was isolated and purified using a filter paper column. The purity of kappa preparations thus obtained was significantly improved by adding a preliminary deciliation step.

Synthesis and release of NAD(P)ase by Neurospora crassa wild type was studied in experiments in which mycelia grown in Vogel minimal medium were transferred to media containing protein as the only carbon source. Several results are presented suggesting that the NAD(P)ase may be induced by the presence of protein in the culture medium. Low concentrations of sucrose or glucose (0·1%), Casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed the enzyme synthesis. Under induction conditions NAD(P)ase and alkaline protease appeared together in the culture medium. It would appear that NAD(P)ase and alkaline protease are coordinately regulated by a common control mechanism related to carbon catabolism.

Gas vesicle protein may account for more than 6% of the cell dry weight and over 10% of the total cell protein in the planktonic cyanobacterium Anabaena Jlos-aquae. The gas vesicles collapsed by rising cell turgor pressure during buoyancy regulation can therefore represent a significant protein pool within this organism. We have looked for evidence of gas vesicle protein recycling. Gas vesicles were isolated from cells of A.80s-aquae that had been pressurized and then cultured in a radioactively labelled medium. The new gas vesicles that formed were less highly labelled than the new vesicles formedin cells from a second culture that had not been pressurized. The results suggest that the collapsed gas vesicles are disaggregated and that the constituent gas vesicle protein is reassembled into new gas vesicles. Proof of this will require experiments using in vitro assembly systems.