The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

An Escherichia coli S1-like ribosomal protein is immunologically conserved in Gram-negative bacteria, but not in Gram-positive bacteria

Abstract A study was made of the enzymology of primary and intermediary pathways of C₁ metabolism in three strains of non-motile obligately methylotrophic bacteria. Each uses a variant of the ribulosemonophosphate (RMP) cycle of formaldehyde fixation which involves the Entner-Doudoroff route for hexose-phosphate cleavage and transaldolase/transketolase mode of rearrangement. The organisms possess high levels of hexulose-phosphate synthase and NAD(P)-linked glucose-6-phosphate and 6-phosphogluconate dehydrogenases. In addition they contain small activities of dye-linked methanol and methylamine dehydrogenases, PMS- and NAD-linked formaldehyde and formate dehydrogenases. This indicates cyclic rather than direct oxidation of formaldehyde derived from methanol or methylamine. The tricarboxylic acid cycle is defective in 2-ketoglutarate dehydrogenase and the glyoxylate shunt is not operating because of the absence of malate synthase. Oxaloacetate is regenerated by (phosphoenol) pyruvate carboxylases. NH⁺ ₄ is assimilated mainly by glutamate dehydrogenase. The results show metabolic similarities between motile and non-motile obligate methanol and methylamine utilizers.     

Control of methionine biosynthesis in Escherichia coli by proteolysis

Most bacterial proteins are stable, with half-lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis - homoserine trans-succinylase (HTS) - is unstable and subject to energy-dependent proteolysis. The enzyme is stable in triple mutants defective in Lon-, HslVU- and ClpP-dependent proteases. The instability of the protein is determined by the amino-terminal part of the protein, and its removal or substitution by the N-terminal part of beta-galactosidase confers stability. The effect of the amino-terminal segment is not caused by the N-end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half-life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.     

Cytokine-stimulating activity of lipopolysaccharide of gram-negative bacteria and its role in antitumor immunity

The review deals with the discussion of different mechanisms of the antitumor action of lipopolysaccharides (LPS), including the induction of endogenous cell mediators, the development of inflammatory and immune response and systemic metabolic changes. The complex action of these factors on the levels of cells, tissues, organs and the whole body led, in some cases, to the suppression or hemorrhagic necrosis of the tumor. These cases were regarded as analogous to the development of the septic syndrome when a cascade of reactions leading to the resorption of tumors was initiated and to chronic inflammation resulting, on the contrary, in the formation of pretumor status. The data on the role of cytokines in carcinogenesis are presented and the possible causes of established contradictions are discussed. The prospects of using LPS in the therapy of oncological diseases are discussed.     

Thymidylate synthase gene expression is stimulated by some (but not all) introns.

We previously described the construction of an intronless mouse thymidylate synthase (TS) minigene that has the normal 5' and 3' flanking regions of the gene linked to full length TS cDNA. Transfection of the minigene into ts- hamster V79 cells led to low level expression of normal mouse TS mRNA and protein. In the present study we analyzed the effect of introns on the expression of the TS minigene in transient transfection assays. Inclusion of introns 5 and 6 at their normal locations in the coding region led to an 8-9-fold stimulation of the level of TS and TS mRNA. Almost all of introns 5 and 6 could be deleted without diminishing the stimulatory effect. Inclusion of intron 3 also stimulated the expression of the minigene, although to a lesser extent than introns 5 and 6. However, inclusion of intron 4 had no stimulatory effect. Analysis of minigenes that contained various combinations of introns revealed that the stimulatory effects of the introns were not additive.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

Targeting LPS biosynthesis and transport in gram-negative bacteria in the era of multi-drug resistance

Gram-negative bacteria pose a major threat to human health in an era fraught with multi-drug resistant bacterial infections. Despite extensive drug discovery campaigns over the past decades, no new antibiotic target class effective against gram-negative bacteria has become available to patients since the advent of the carbapenems in 1985. Antibiotic discovery efforts against gram-negative bacteria have been hampered by limited intracellular accumulation of xenobiotics, in large part due to the impermeable cell envelope comprising lipopolysaccharide (LPS) in the outer leaflet of the outer membrane, as well as a panoply of efflux pumps. The biosynthesis and transport of LPS are essential to the viability and virulence of most gram-negative bacteria. Thus, both LPS biosynthesis and transport are attractive pathways to target therapeutically. In this review, we summarize the LPS biosynthesis and transport pathways and discuss efforts to find small molecule inhibitors against targets within these pathways.     

Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents.

The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium.     

Complement activation via the alternative pathway by purified Salmonella lipopolysaccharide is affected by its structure but not its O-antigen length

Salmonellae, the lipopolysaccharide of which differ in the chemical structure of their O-antigenic side chains, were previously shown to activate C3 at differential rates via the alternative pathway. We wanted to test whether lipopolysaccharide isolated from these strains yields identical results, and also the effect of the polysaccharide chain length, which varies from 0 to 40 or more repeating units in a single strain. Lipopolysaccharide was purified from the above strains, hydrolyzed (0.1 N NaOH, 56 degrees C, 30 min), and used to coat sheep erythrocytes to different densities, and C3 activation in C4-deficient guinea pig serum was measured. C3 activation was proportional to lipopolysaccharide density and time, and the relative rates and extents of activation by this bacteria-free system were the same as for the original bacteria. Activation was reduced 10 to 15% when the serum was preabsorbed with strains either containing or lacking O-antigen side chain, suggesting augmentation by antibody; however, even after multiple absorptions, activation varied with O-antigen structure as expected. This differential activation was not due to differences in the average length of the O-antigenic polysaccharide chains, because the size was similar for all three lipopolysaccharides. Moreover, the extent of activation by lipopolysaccharide that had been fractionated on a column of Sephadex G-200 was independent of the polysaccharide chain length for lengths greater than 3 repeating units. The results prove that C3 activation by lipopolysaccharide via the alternative pathway is sensitive to slight variations in the chemical structure, but not to large variations in length of the O-antigen polysaccharide side chain of lipopolysaccharide.     

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.