- Volume 132, Issue 3, 1986

SUMMARY: The protein content of culture supernatants of three Erwinia chrysanthemi strains, B374, 3937j and 3665, grown on different carbon sources was compared. After growth in presence of polygalacturonate, four new polypeptides, identified as pectinases, were synthesized. These induced proteins, and the pattern of pectate lyase induction, differed among the strains. The proteins present in the supernatants of some mutants known or suspected to be affected in pectinase production (secretion-defective mutants and mutants in the degradative pathway of galacturonate and ketodeoxygluconate) were also analysed.

SUMMARY: The kinetics of maturation of certain exported proteins were analysed in Escherichia coli strains that also concomitantly overproduce either a periplasmic protein or the leader peptidase. The results led to three conclusions. (a) Overproduction of leader peptidase has no effect on the rate of maturation of at least two exported proteins, one periplasmic (TEM β-lactamase), one outer membrane (PhoE); therefore, the quantity of leader peptidase is not rate-limiting for normal export. (b) Overproduction of PhoS reduces the rate of maturation of two other periplasmic proteins (β-lactamase and PhoA) and itself, presumably by competing for the rate-limiting component of the export apparatus. (c) Overproduction of leader peptidase in a strain overproducing PhoS has no effect on the retarded maturation of PhoS. Therefore even in these conditions, leader peptidase is not rate limiting.

SUMMARY: The 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolase encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2 (PaWl) was purified to homogeneity. It has an M r of 65000 and is dissociated by SDS into two subunits of equal size. Alanine was the only N-terminal residue detected, and each subunit contained one cysteine thiol group. The pH optimum for activity and for enzyme stability was around 7·5, whereas the isoelectric point was 4·7. Only the products of catechol 2,3-oxygenase action on linear chain alkylcatechols and 4-chlorocatechol served as substrates. Kinetic measurements showed that the higher activity against the ketone substrates (from 3-substituted catechols) over aldehyde substrates (from 4-substituted catechols) was the result of higher V max values rather than lower K m values. Antisera prepared against this purified HOD hydrolase were shown by Ouchterlony double diffusion and inhibition studies to be related to the HOD hydrolase from phenol-grown P. putida strain U (NCIB 10015) in which the enzyme is chromosomally encoded.

SUMMARY: Two forms of citrate synthase (EC 4.1.3.7) have been found in several species of Pseudomonas, a ‘large’ form (M r ≃ 250000) which is generally inhibited by NADH and reactivated by AMP, and a ‘small’ form (M r ≃ 100000) which is insensitive to these nucleotide effectors. Other species of Pseudomonas were found to contain either the ‘large’ or the ‘small’ form. Gel filtration and ion-exchange with the technique of fast protein liquid chromatography were used to resolve the enzymes. Where both citrate synthases were present, there did not appear to be an equilibrium between the two forms. The results reveal a new and complex diversity of citrate synthase within the genus Pseudomonas.

SUMMARY: Extracellular polysaccharide (EP) and lipopolysaccharide (LPS) were isolated from three strains of Pseudomonas fluorescens (a wild-type and two mutants) and the adsorption isotherms (relationship between amount of polymer adsorbed and bulk liquid concentration of polymer in solution) of these polymers to hydrophobic, tissue culture treated and sulphonated polystyrene surfaces were measured. The adsorption properties of the polymers were then related to the ability of the three bacterial strains to attach to the polystyrene surfaces in an attempt to elucidate the attachment mechanisms. A Langmuir adsorption isotherm equation was applied to the data, and the mathematical constants thus derived indicated if and at what concentration each surface became polymer-saturated and whether multilayer adsorption occurred. EP isolated from a crenated mutant (strain with the greatest attachment ability) adsorbed at higher concentrations than EP from wild-type and mucoid strains, and the isotherm indicated multilayer adsorption. EP from the mucoid strain (strain with little attachment ability) showed comparatively little adsorption. The isotherm of wild-type LPS was very similar to that of EP from the mucoid strain. Polymer adsorption to the three surface types was different and was generally consistent with the different degrees of bacterial attachment to the surfaces.

SUMMARY: During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML 308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh- Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.

SUMMARY: Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced, protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.

SUMMARY: The somatic incompatibility reaction occurring at sites of fusion between hyphae of genetically different secondary mycelia of Phanerochaete velutina has been examined using combined light and electron microscopy. Hyphal compartments affected by incompatibility rapidly showed increased vacuolation and the development of autophagic bodies throughout the cytoplasm. Dense osmiophilic spherical bodies that developed within the vacuole lumen characterized the early and highly regulated phase of the reaction. Eventually, as expansion of the vacuolar system proceeded, more widespread degeneration began in the remaining cytoplasm, nuclei and mitochondria. The large microtubule bundles present within this species showed variable behaviour during degeneration of the cell compartments, either breaking down early on in the process or persisting intact during breakdown of the other cell components. The incompatibility finally caused extensive disruption and death of the compartments engaged in fusion and often contiguous cells. Plugging of dolipore septa apparently restricted spread of the incompatibility response along the fused hyphae.

SUMMARY: The localized cell wall lysis associated with asexual spore release from sporangia and spore cysts of saprolegniaceous water moulds appears, based on immunocytochemical and inhibitor studies, to be dependent on calmodulin (CaM). Distinct bands of CaM surround the exit pores of sporangia and cysts produced by Achlya ambisexualis, Dictyuchus monosporus and Saprolegnia ferax. In differentiating sporangia and cysts, CaM becomes concentrated in the apical papillae, at the tips of which exit pores are formed. While all stages of sporulation can be inhibited to an extent by micromolar concentrations of trifluoperazine, an anti-CaM drug, spore release is the most sensitive.

SUMMARY: The localization of ATP-hydrolysing activity in vegetative cells, spores and isolated membranes of Bacillus subtilis 168 was studied by a cytochemical method combined with electron microscopy. The activity was located mainly in the cytoplasmic membrane and the mesosomes, and was also found in the inner layer of the cell wall facing the cytoplasmic membrane. Activity was also detected in the cross-membranes of dividing cells and in spore coats. The product of the reaction was observed either as fine electron-dense granules incorporated into the membranes, or as high-contrast lead precipitates on the surfaces of the membranes.

SUMMARY: We have inserted a C-terminally truncated gene of the major outer membrane protein ompA of Escherichia coli downstream from the promoter and signal sequence of the secretory α-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the α-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of ompA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.

SUMMARY: The nucleotide sequence of a 286 bp fragment containing the uxaB control region of Escherichia coli has been determined. The transcriptional start of the uxaB gene has been located and the promoter signals identified. Various fragments of the uxaB promoter-proximal region were fused in vitro with the lacZ gene. Results obtained with these fusions indicate that the operator-promoter sites are located on a 110 bp restriction fragment. The determination of the amino acid sequence of the NH2-terminus of the uxaB gene product revealed that the uxaB gene is not initiated with the AUG codon but with the unusual GTG codon. CRP, the cyclic AMP receptor protein, does not bind to the uxaB control region DNA even though expression of the uxaB gene is sensitive to catabolite repression.

SUMMARY: The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.

SUMMARY: The development of an optimized in vitro polyuridylic acid-dependent polyphenylalanine-synthesizing system using cell-free extracts of the basidiomycete fungus Coprinus cinereus is described. The in vitro assay has been used to show that cycloheximide-resistant strains CY8.2, CY9.23 and Sp98, all mutant at the cy-2 locus, have cytoplasmic ribosomes which are more resistant to the drug than the corresponding sensitive strains, CY8, CY9 and CY3. Cycloheximide concentrations and molar ratios of cycloheximide to ribosomes required for 50% inhibition in vitro under standard assay conditions are presented for these strains. The molar ratio required for 50% inhibition in vitro is dependent on the concentration of ribosomes in the assay.

SUMMARY: The DNA-intercalating agent ethidium bromide was used to eliminate plasmid DNA from streptomycetes. Other mutational events associated with chromosome changes occurred at high frequency; they resulted in phenotypic changes such as loss of enzyme activity, antibiotic resistance or ability to sporulate.

SUMMARY: Bacteriophages which mediate the conversion of the O somatic antigen of Salmonella choleraesuis from the 627 to the 617 phenotype have been isolated from two strains of S. newport and one of S. muenchen, and also from sewage collected from two areas where there have been no reports of S. cholerae-suis infection for several years. The phages differed from each other by cross-resistance tests.