Influence of the cell envelope on the accumulation of cadmium by a marine pseudomonad

The accumulation of cadmium by a sensitive marine pseudomonad was investigated on the basis of the physiological state of its cell envelope. The more impaired the envelope the greater the accumulation. This phenomenon was due to a greater number of adsorption sites for cadmium on the inner layers and/or to the impermeability of the cell envelope.     

Proteins of the cell envelope of a marine pseudomonad, Pseudomonas BAL-31

The protein composition of the envelope fraction of Pseudomonas BAL-31 was studied by polyacrylamide gel electrophoresis. Two major polypeptides of molecular weights 130 000 and 110 000 were found. These two polypeptides, which account for as much as 40–50% of the total protein of the envelope, are associated with the outer membrane. One of these proteins might be a glycoprotein. The inner membrane contains a more heterogeneous collection of smaller polypeptides.     

Characterization of neutral amino acid transport in a marine pseudomonad.

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Cation interactions and biochemical composition of the cell envelope of a marine bacterium

Envelopes of a marine isolate, c-A1, and of a terrestrial isolate, 121, were compared for their susceptibility to disintegration in distilled water after exposure to 0.05 m MgCl(2) and to 0.1 and 1.0 m NaCl. After exposure to MgCl(2) alone, both types of envelopes remained intact in distilled water. Envelopes of marine isolate c-A1, but not of the terrestrial isolate, fragmented in distilled water after exposure to 1.0 m NaCl. Partial reaggregation of the c-A1 envelope fragments occurred on addition of MgCl(2). In cation-exchange experiments, bound Mg(++) in the envelopes of both organisms was displaced by Na(+). The envelopes of c-A1 were found to contain lipopolysaccharide, muramic acid, and a variety of phospholipids, of which the major component was phosphatidylethanolamine, accompanied by lesser amounts of phosphatidic acid, diphosphatidylglycerol, and phosphatidylserine. Analyses of envelope acid hydrolysates revealed a similar amino acid distribution in the marine and terrestrial isolates, but envelopes of c-A1 had less than half the total amino acid content of envelopes of 121 per envelope dry weight. Possible relationships between cations and biochemical components of the envelopes are considered in terms of differences in behavior of the two organisms in low ionic environments.     

Diminution of outer membrane permeability by Mg2+ in a marine pseudomonad.

Intact cells of the marine pseudomonad MB-45, in the presence of optimal Mg2+, exhibited little alkaline phosphatase activity as judged by the hydrolysis of p-nitrophenylphosphate. Sonic extracts, in contrast, were rich in this activity. Removal of the loosely bound outer layer did not diminish this crypticity of alkaline phosphatase, but decreasing the concentration of Mg2+ in the suspending medium progressively exposed the alkaline phosphatase. Since MB-45 did not liberate alkaline phosphatase into the surrounding medium even in the absence of Mg2+ and since this enzyme is localized in the periplasmic space, it can be concluded that the crypticity was due to the exclusion of p-nitrophenylphosphate by the outer membrane. Mg2+ is apparently essential for the full expression of this limited permeability.     

Kinetics of Na+-dependent K+ ion transport in a marine pseudomonad.

The effect of external Na plus concentration on the transport of K plus was studied using K plus-depleted cells of a marine pseudomonad. K plus transport was found to be a saturable process and requires Na plus. The initial rates for K plus transport over a range of external K plus concentrations were measured in suspensions containing various fixed concentrations of Na plus. Reciprocals of the initial rates for K plus transport were plotted against reciprocals of the external concentration of K plus or Na plus to yield two primary Lineweaver-Burk plots. The experimental data were found to fit bisubstrate enzyme kinetics, with a sequential type mechanism. However, the initial rate data did not allow distinction between ordered or random mechanisms. The results suggest that Na plus and K plus form a ternary complex with a specific K plus carrier molecule on the outer surface of the membrane prior to translocation and the release of K plus inside the cell.     

K plus-dependent deplasmolysis of a marine pseudomonad plasmolyzed in a hypotonic solution

When cells of a marine pseudomonad were washed with a solution consisting of 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl (complete salts), they maintained their normal morphology. When washed with a solution of 0.05 m MgSO(4), they became plasmolyzed as indicated by both phase and electron microscopy. Suspensions of cells washed with 0.05 m MgSO(4) showed an increase in optical density (OD) when 0.3 m NaCl was added, and this was followed by a decrease in OD upon the further addition of 0.01 m KCl. Salts of other monovalent cations were not effective in replacing K(+) in producing the OD decrease. Phase-contrast microscopy revealed that the increase in OD was accompanied by a decrease in cell size, and the decrease in OD, by an increase in the cell size. Both phase and electron microscopy showed that the K(+)-dependent decrease in OD was accompanied by deplasmolysis of the cells. Na(+) was required in the suspending medium in addition to K(+) to obtain deplasmolysis. The intracellular K(+) concentration in cells which had been washed with complete salts and which had retained their normal morphology was found to be 0.290 m. In cells plasmolyzed by washing with 0.05 m MgSO(4), the intracellular K(+) concentration was 0.004 m. Deplasmolyzed cells contained 0.330 m K(+). The membrane profile of plasmolyzed cells was retained when protoplasts were formed. The protoplasts became spherical if incubated in a solution permitting the deplasmolysis of the parent cells. The evidence obtained indicates that plasmolysis and deplasmolysis under the conditions described was due to the loss and gain, respectively, of K(+) by the cells. The effect of Na(+) could be ascribed to its capacity to control the porosity of the cytoplasmic membrane of this organism.     

Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium.

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.     

Distribution of mechanical stress in the Escherichia coli cell envelope

The cell envelope in Gram-negative bacteria comprises two distinct membranes with a cell wall between them. There has been a growing interest in understanding the mechanical adaptation of this cell envelope to the osmotic pressure (or turgor pressure), which is generated by the difference in the concentration of solutes between the cytoplasm and the external environment. However, it remains unexplored how the cell wall, the inner membrane (IM), and the outer membrane (OM) effectively protect the cell from this pressure by bearing the resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis. In this study, we have used molecular dynamics (MD) simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension. We calculated the area compressibility modulus of all three components in simulations from tension-area isotherms. Experiments on monolayers mimicking individual leaflets of the IM and OM were also used to characterize their compressibility. While the membranes become softer as they expand, the cell wall exhibits significant strain stiffening at moderate to high tensions. We integrate these results into a model of the cell envelope in which the OM and cell wall share the tension at low turgor pressure (0.3 atm) but the tension in the cell wall dominates at high values (>1 atm).