Edward R. Leadbetter

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.

Metabolism of sulfonic acids and other organosulfur compounds by sulfate‐reducing bacteria

This article presents a short review of recent research that established the ability of sulfate‐reducing bacteria to utilize sulfonic acids as terminal electron acceptors (TEA) for anaerobic respiratory growth. Newer studies of the bacterium most intensively investigated, Desulfovibrio desulfuricans, strain ICI, are also reported. When either of two sulfonic acids examined—isethionate (2‐hydroxyethanesulfonate) or cysteate (alanine‐3‐sulfonate)—served as sole TEA, key changes in the cells’ enzymo‐logical profile occurred: decreased production of two enzymes involved in sulfate reduction, namely, ATP sulfurylase and APS reductase. Similar reduction in content of these enzymes was seen when either sulfite or fumarate served as TEA. Protein profiles (polyacrylamide gel electrophoresis) of extracts of cells grown with different TEA revealed the presence of a 97‐kD polypeptide apparently unique to isethionate‐grown cells; a different polypeptide was noted in extracts of cysteate‐grown cells. The absence of such stained bands in extracts of sulfate‐grown cells suggests that these polypeptides are involved in utilization of sulfonic acids as TEA. H2 threshold values of cells growth with isethionate as TEA were significantly lower than for cells growing with sulfate or sulfite, suggesting that energy may be conserved in the cleavage of isethionates C‐S linkage. A survey of the distribution of sulfonic acids in diverse habitats combined with the ability of other anaerobic bacteria to respire these compounds leads to the suggestion sulfonate reduction is likely to be significant in the sulfur cycle.

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.

Isolation and characterization of a Chryseobacterium strain from the gut of the American cockroach, Periplaneta americana

Abstract. A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium. In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect. Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes. Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype. This phenotypes abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight). The highest population density was in the hindgut, ranging from 2×106 to 1.2×107 colony forming units ml–1 during a 1-year period. The nature of the symbiosis between the FR2 phenotype and P. americana is discussed.

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.

Surface-induced synthesis of new sulfonolipids in the gliding bacterium Cytophaga johnsonae

Many simple gliding bacteria contain significant quantities of phosphate-free, sulfur-containing lipids (sulfonolipids; N-acylamino-3-hydroxyisoheptadecane-1-sulfonic acids, or N-acyl capnines) that recently were shown to function in the ability of Cytophaga johnsonae to migrate over solid surfaces. Reported here is the synthesis, by surface-grown Cytophaga johnsonae cells, of two additional sulfonolipids not present in cells grown in liquid media. These newly characterized sulfonolipids are more polar than the N-acylcapnines characteristic of liquid grown cells. Acid methanolysis of the sulfonolipids revealed that the aminosulfonate capnine was common to all, thus indicating that the chemical differences in the compounds resided in their N-fatty acyl groups, and not in the aminosulfonate moiety. Instead of the non-hydroxy and 3-hydroxy fatty acyl moieties present in sulfonolipids of liquid-grown cells, one new sulfonolipid contained a 2-hydroxy, branched C15 fatty acid, while the other contained a 2,3-dihydroxy, isobranched C16 fatty acid, as indicated by gas chromatographic and mass spectrometric analyses. Although the structure of sulfonolipids thus varies between surface- and liquid-grown cells, no difference was found between the total quantity of sulfonolipids present under either of these conditions. The surface-dependent synthesis of these more polar N-acyl-aminosulfonates ceased immediately when surface-grown populations were suspended in broth. The ability of Cytophaga johnsonae to synthesize these compounds in response to a solid surface may be significant in relation to the organisms ability to migrate over such surfaces; it is one of few instances where a physical interaction of the cell surface has been shown to influence the molecular composition of a prokaryote.

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.