Walter Godchaux

Sulfonates : novel electron acceptors in anaerobic respiration

Abstract The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate.

Sulphonolipids are molecular determinants of gliding motility

A variety of bacteria1 that possess no obvious locomotor organelles are, nonetheless, able to translocate on solid surfaces (but not through liquids). In no case has the mechanism of this ‘gliding’ motility been elucidated2. Our recent discovery that unusual sulphonolipids3,4 (Fig. 1) are major cell-envelope components peculiar to the Cytophaga-Flexibacter group of gliding bacteria5 led us to examine whether these lipids are important in this motility. Mutants deficient in both gliding and sulphonolipid synthesis were isolated from mutagenized cultures of Cytophaga johnsonae (our laboratory strain, originally ATCC 17061). In some of these mutants, restoration of sulphonolipid content through provision of a specific biosynthetic precursor resulted in recovery of the ability to glide. The sulphonolipids are the first molecules shown to be specifically required for gliding motility (surface translocation).

Sulphonate utilization by enteric bacteria.

A variety of sulphonates were tested for their ability to serve as nutrients for Escherichia coli, Enterobacter aerogenes and Serratia marcescens. Cysteate, taurine and isethionate could not serve as sole sources of carbon and energy but, under aerobic conditions, could be utilized as sources of sulphur. Both sulphate and sulphonate supported equivalent cell yields, but the generation times varied with the sulphonate being metabolized. The sulphonate-S of HEPES buffer, dodecane sulphonate and methane sulphonate was also utilized by some strains, whereas the sulphonate-S of taurocholate was not. None of the sulphonates tested served as a sulphur source for growth under anaerobic conditions. Sulphonate utilization appears to be a constitutive trait; surprisingly, however, cells of E. coli and Ent. aerogenes utilized sulphate-S in preference to that of sulphonate, when both were present. E. coli mutants unable to use sulphate as a source of sulphur because of deficiencies in sulphate permease, ATP sulphurylase, adenylylsulphate kinase (APS kinase) or glutaredoxin and thioredoxin were able to utilize sulphonates; hence sulphate is not an obligatory intermediate in sulphonate utilization. However, mutants deficient in sulphite reductase were unable to utilize sulphonates; therefore, this enzyme must be involved in sulphonate utilization, though it is not yet known whether it acts upon the sulphonates themselves or upon the inorganic sulphite derived from them.

Utilization of sulfonates as sole sulfur source by soil bacteria including Comamonas acidovorans

Bacteria able to use cysteate, taurine or isethionate as sole source of carbon and energy were isolated from the soil. Tests of sulfur assimilation showed that sulfonate sulfur and sulfate sulfur supported comparable cell yields. Methanesulfonate, 1-dodecanesulfonate and p-toluenesulfonate also served as sole source of sulfur for strain I91, identified as Comamonas (Pseudomonas) acidovorans. Competition studies with strain I91 showed that the presence of sulfate inhibits cysteate, isethionate or taurine incorporation. Pseudomonas aeruginosa PAO1, Comamonas acidovorans 14 and 105, and Acidovorax (Pseudomonas) facilis 332 used cysteate, isethionate, or taurine as sole source of sulfur while P. aeruginosa PAO716 and PAO718 used only taurine.

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.

Further studies on the inhibition of cellular protein synthesis by vesicular stomatitis virus

Abstract After infection of L cells by vesicular stomatitis virus, the rate of protein synthesis steadily declines. Simultaneously, polysomes disaggregate and 80 S ribosomes accumulate. Transit time studies indicate that the average rate of polypeptide chain elongation is virtually identical for infected and uninfected cells. Cellular mRNAs remain intact and potentially functional in infected cells, as determined by their ability to be translated into cellular proteins in nuclease-treated reticulocyte lysates. More template activity is present in infected cells than in uninfected cells as a result of the presence of viral mRNAs in infected cells. Extracts from infected L cells translate their endogenous mRNAs less efficiently than do comparable extracts from uninfected cells. Nuclease-treated extracts from infected cells also show a lesser ability to translate given amounts of exogenous L cell or vesicular stomatitis virus mRNAs than do comparable extracts from uninfected cells. This observation suggests that the translation of both cellular and viral mRNAs is affected equally by the lesion induced in the cell by virus infection. The lesion appears to act at the level of initiation of protein synthesis and results in an underutilization of the total template activity present in infected cells.

Diversity in surface features of Cytophaga johnsonae motility mutants

Twenty-eight non-spreading mutants of the gliding bacterium Cytophaga johnsonae were generated by different approaches and subsequently studied to determine whether they possessed traits commonly assumed to be associated with such mutants. Many non-spreading variants of C. johnsonae purportedly possess a static cell surface as opposed to the dynamic one of the motile parent strain. A moving cell surface has been supposed to be responsible for an array of traits associated with motile cells, but missing in non-motile cells. Phage sensitivity, chitin digestion and the ability of cells to move latex beads over their surfaces are some of the alleged motility-dependent traits. Also, it has been reported that non-spreading mutants possess a cell surface that is less hydrophobic than that of the parent strain. We characterized our collection of mutants in relation to the above mentioned traits to determine whether these characteristics indeed required a moving cell surface. Our findings showed that neither phage sensitivity nor chitin digestion were motility-dependent. In addition we noted that non-spreading mutants could possess surfaces more (rather than less) hydrophobic than the motile parent strain.

Biosynthesis of a sulfonolipid in gliding bacteria

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.

Acetate acts as a protonophore and differentially affects bead movement and cell migration of the gliding bacterium Cytophaga johnsonae (Flavobacterium johnsoniae)

Cells of Cytophaga johnsonae (now Flavobacterium johnsoniae) are able to translocate on solid surfaces but are unable to swim in liquid media. Organelles that may be involved in this gliding motility have not been detected, and the mechanism(s) responsible remains unknown. The movement of latex beads attached to the cell surface is considered by some to be a manifestation of the gliding machinery. In this study, acetate (in nutrient-level quantity, 45 mM) was found to inhibit bead movement on cell surfaces, whilst formation and movement of groups of cells (rafts) and typical colony spread were not affected; generation time (in liquid culture) was only slightly increased. Since acetate is a weak acid and is recognized as a protonophore, various electron-transport-associated features were assessed in an effort to understand the differential effects of acetate on bead movement and cell motility. Selected protonophores and electron transport inhibitors were tested to compare their effects on cell translocation and metabolic activities with those of acetate. Although O2 consumption was not significantly affected in the presence of acetate and the protonmotive force decreased only minimally, ATP levels were markedly decreased. Arsenate and cyanide were also shown to inhibit bead movement but did not inhibit either movement of rafts of cells or colony spreading. Cyanide lowered O2 consumption, while arsenate did not; both compounds effected substantial decreases in cellular ATP content, but little or no decrease in protonmotive force. The inhibitory effects of these compounds on bead movement over cell surfaces contrasted with the continued ability of cells to form rafts, to glide and to form spreading colonies and led to the conclusion that bead movement is not a complete correlate of the gliding machinery of C. johnsonae. In addition, it seems likely that bead movement is more affected by the level of cellular ATP than it is by the protonmotive force, which has been assumed to provide the energy (derived from the transmembrane gradients) for the gliding machinery.