Phospholipids and a novel glycine-containing lipoamino acid in Cytophaga johnsonae Stanier strain C21.

Two-dimensional thin-layer chromatography revealed that Cytophaga johnsonae contains at least 10 kinds of lipid, 2 of which are phospholipids, namely, phosphatidylethanolamine and phosphatidylcholine. One of the remaining lipids is a novel lipid that contains an amino acid. The structure of this unusual lipid (lipoamino acid) was resolved by chemical and physicochemical methods. The fatty acyl moiety of this lipid was diverse. The structure of the major molecular species of the lipid was determined as iso-3-hydroxy heptadecanoic acid, amide linked to glycine and esterified to isopentadecanoic acid. This type of glycine-containing lipid is a novel biological material which we have called cytolipin, basing this nomenclature on the genus of the bacterium. This is the first report of the lipid composition of C. johnsonae.

Use of nonmotile mutants to identify a set of membrane proteins related to gliding motility in Cytophaga johnsonae.

Nonmotile mutants of the gliding bacterium Cytophaga johnsonae were examined to identify proteins that might be involved in gliding motility. Wild-type and mutant cell proteins were solubilized and fractionated by using Triton X-114, and the proteins that partitioned into the aqueous phase or the detergent phase were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for proteins that differed between wild-type and mutant cells. Seventeen proteins, ranging in size from 16 to 150 kilodaltons, were implicated by this technique as having some relationship to gliding and were designated Gld-1 through Gld-17. All Gld proteins behaved as integral membrane proteins, partitioning into the detergent phase. All 56 mutants examined exhibited changes in 1 or more of the Gld proteins, with the number of proteins altered in any mutant varying from 1 to 11. Several lines of evidence suggested that proteins Gld-12 through Gld-15 are glycoproteins. Analysis of banding patterns of detergent-fraction proteins of motile revertants supported the idea that the Gld proteins have a role in gliding motility.

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 and its mutants defective in adhesion and motility.

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen). At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion. The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight. An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern. Two other Adh2 revertants resembled their adhesion-defective parent. The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type.

Effect of temperature shifts on gliding motility, adhesion, and fatty acid composition of Cytophaga sp. strain U67.

Gliding motility and flipping of 25 degrees C-adapted Cytophaga sp. strain U67 were inhibited when the bacteria were shifted to a less than or equal to 12 degrees C environment; motility was not blocked by a shift to 13 degrees C. Bacteria adapted to 4 degrees C were motile over the entire 4 to 25 degrees C temperature range tested. U67 adhesion to the substratum appeared to be unaffected by temperature shifts. Bacteria adapted to 4 degrees C had higher proportions of unsaturated and branched-chain fatty acids than did those grown at 25 degrees C. When 25 degrees C-adapted bacteria were subjected to a gradual temperature decline, the time of reappearance of gliding competence at 4 to 5 degrees C was correlated with these changes in fatty acid composition.

Topostin, a novel inhibitor of mammalian DNA topoisomerase I from Flexibacter topostinus sp. nov. II. Purification and some properties of topostin.

We describe the isolation of inhibitors of mammalian DNA topoisomerase I, named topostins, from a culture broth of Flexibacter topostinus sp. nov. and some properties of the inhibitors. Topostins A1, A2 and B were isolated by differential solubility in solvents, adsorption chromatography on silica gel and gel filtration on a Sephadex LH-20 column. Topostins A1, A2 and B had specific activities of 4,700, 16,000 and 22,000 U/mg, respectively. The most active metabolite topostin B comprised two components with MW of 567 and 553 in an equimolar ratio.

PB-5266 A, B and C, new monobactams. I. Taxonomy, fermentation and isolation.

New monobactams, PB-5266 A, B and C were isolated from the culture filtrate of Cytophaga johnsonae PB-5266 by various types of column chromatography and preparative reverse phase HPLC. PB-5266 A, B and C exhibited weak antibacterial activity against a sensitive mutant of Escherichia coli to beta-lactam antibiotics.

PB-5266 A, B and C, new monobactams. II. Physico-chemical properties and chemical structures.

The chemical structures of three new monobactams, PB-5266 A, B and C, were elucidated by their physico-chemical properties and spectrometric studies. In contrast to previously described monobactams, they all possess a dehydroasparagine residue.

Properties of Cytophaga johnsonae strains causing spoilage of fresh produce at food markets.

Two strains of gliding, orange-pigmented bacteria, isolated from fresh bell pepper and watermelon, respectively, showing soft-rot lesions, were identified as Cytophaga johnsonae. They differed from seven type strains of C. johnsonae deposited at the American Type Culture Collection (ATCC) in some properties, such as the ability to utilize glucose, xylose, trehalose, rhamnose, and sucrose. Spherical bodies resembling microcysts of Sporocytophaga sp. in addition to short rods and long filaments were observed in two strains (ATCC 29583 and 29588) throughout the growth cycle and also in aged cultures of other strains. All strains examined were shown to degrade five natural or synthetic polymers (pectin, chitin, starch, protein, and carboxymethyl cellulose). Only six strains (including ATCC 17061, 29587, 29589, and 19366) were able to infect and macerate artificially wounded potato tubers and fruits of pepper, squash, and tomato. The pathogenic strains secreted more pectate lyase in broth medium than the nonpathogenic strains. C. johnsonae, generally known as a soil saprophyte, might occasionally act as an opportunistic pathogen, causing decay of fresh produce in storage or in transit.

Isolation and characterization of a new Cytophaga species implicated in a work-related lung disease.

A yellow-pigmented, gram-negative, gliding bacterium isolated from an industrial water spray air humidification system was implicated as a causative agent in several occurrences of lung disease with hypersensitivity pneumonitis-like symptoms. The bacterium, designated WF-164, lacked microcysts or fruiting bodies and had a DNA base composition of 34.8 mol% of guanine plus cytosine. Gliding, flexing, nonflagellated cells measuring 0.3 by 3.5 to 8.9 micron were observed by using light and electron microscopy. Tests to determine utilization of selected carbohydrates revealed an amylolitic, chitinoclastic, noncellulytic bacterium. A number of additional biochemical and physiological tests were performed. DNA homology studies detected a 77.8% similarity to Cytophaga aquatilis (ATCC 29551). Comparisons of cellular fatty acid and carbohydrate contents of isolate WF-164 with a Flexibacter sp., several Cytophaga spp., and Flavobacterium reference strains revealed similar patterns to that of C. aquatilis. On the basis of these characteristics, isolate WF-164 was identified as a new Cytophaga sp.

Sulfonolipids of gliding bacteria. Structure of the N-acylaminosulfonates.

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.

Unusual sulfonolipids are characteristic of the Cytophaga-Flexibacter group.

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.

Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains.

Nine menaquinone-forming strains of the Flavobacterium--Cytophaga complex with DNA base compositions between 35 and 45 moles percent guanine-plus-cytosine were investigated for genome sizes and DNA relatedness by DNA:DNA hybridization in vitro, using the optically recorded initial reassociation kinetics. Two strains representing C. hutchinsonii and C. marinoflava proved to be related on the 50 percent binding level, i.e. on a level of DNA relatedness commonly found within well-classified conventional genera of bacteria. Strains of C. johnsonae, F. heparinum, F. meningosepticum, F. odoratum, F. pectinovorum, and an unnamed Flavobacterium--Cytophaga strain were found to be interrelated, and linked to the genus Cytophaga, on the 30, or 20 percent binding levels, respectively. These findings indicate that the organisms in question are related to Cytophaga. They therefore should be transferred into the family Cytophagaceae.

Iso-branched 2- and 3-hydroxy fatty acids as characteristic lipid constituents of some gliding bacteria.

The fatty acids present in the total hydrolysates of several gliding bacteria (Myxococcus fulvus, Stigmatella aurantiaca, Cytophaga johnsonae, Cytophaga sp. strain samoa and Flexibacter elegans) were analyzed by combined gas-liquid chromatography and mass spectrometry. In addition to 13-methyl-tetradecanoic acid, 15-methyl-hexadecanoic acid, hexadecanoic acid, and hexadecenoic acid, 2- and 3-hydroxy fatty acids comprised up to 50% of the total fatty acids. The majority was odd-numbered and iso-branched. Small amounts of even-numbered and unbranched fatty acids were also present. Whereas 2-hydroxy-15-methyl hexadecanoic acid was characteristic for myxobacteria, 2-hydroxy-13-methyl-tetradecanoic acid, 3-hydroxy-13-methyl-tetradecanoic acid, and 3-hydroxy-15-methyl-hexadecanoic acid were dominant in the Cytophaga-Flexibacter group.

Conformations of A,T-rich DNAs.

DNAs from the genomes of Clostridium perfringens and Cytophaga johnsonii display orthodox A-DNA and B-DNA structures despite their high (A+L) nucleotide content. Unique structures, such as those found for synthetic DNAs having specific special sequences, do therefore not necessarily occur for DNAs having more random base sequence even if these have unusual base compositions. Clostridium perfringens DNA exhibits unusual structural properties only prior to purification by gel filtration.

Direct evidence of the C-like form of sodium deoxyribonucleate.

From infrared linear dichroism studies, direct evidence is given for a new Na DNA form, which is similar to the C form of Li DNA. This C-like form in Na DNA occurs under conditions of very low NaCl content (at high relative humidity), high NaCl content (at low relative humidity), and low G + C content of the DNA (at low relative humitiy). The C form of Li DNA was originally discovered by x-ray diffraction, but has not previously been characterized by infrared spectroscopy. The C form is primarily characterized by the orientation of the O2- -O3 line of the phosphate group, which is about 48 degrees (+/-2 degrees ) with respect to the helical DNA axis and which is different from that of the B and A forms. Na DNA molecules rich in A + T may entirely or partially adopt the C form at low relative humidity. Since from infrared evidence, the C-like form is found not only in Li DNA at low relative humidity but also in Na DNA under different conditions, the C form may be biologically important. A possible role for recognition by proteins is suggested.

Fatty acids of Myxococcus xanthus.

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C(14) to C(17) species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11-28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C(15) and C(17) acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.