Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Heterogeneity of deoxyribonucleic acid molecules isolated from Mycobacterium smegmatis.

Bulk chromosomal deoxyribonucleic acids (DNAs) of Mycobacterium smegmatis strains 607+ (wild type) and 607-1 (Strr) and orange-red pigmented variants (OR) were separated into two distinct bands (types 1 and 2) by cesium chloride density gradient centrifugation. Thermal denaturation analyses showed that type 1 and 2 DNA fragments of these strains possessed guanine plus cytosine contents averaging 69.2% and 60.8%, respectively. Type 1 and 2 DNAs from all strains tested were recovered in relatively equal quantities upon isolation and were found to have similar molecular weights (3.0 x 10(7)). Spectrophotometric assay of DNA reassociation showed that homology between any type 1 and 2 DNA fragments was always very low (29 to 33%), even within the same strain. Homologies among type 1 DNAs isolated from any strain were always high (92 to 98%), whereas homologies between type 2 DNA isolated from OR strains and that from their parental strain 607-1 were lower (51 to 55%). Transformation experiments revealed that methionine, leucine, folic acid, and streptomycin markers were found exclusively in type 1 DNA fragments. In addition to the two types of chromosomal DNA, plasmid DNA possessing a molecular weight of about 4 x 10(6) was found in strain 607-1.     

Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin.

A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves. ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K-12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101. In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.     

Characterization of two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A 190L and their deoxyribonucleic acids

Two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A strain 190L and their deoxyribonucleic acids (DNAs) were purified and characterized. Phage alpha 1, which is unable to form plaques on any strain of C. botulinum, was produced in large quantities after treatment with mitomycin C (MC), whereas phage alpha 2, which was induced in much lower quantities than phage alpha 1, propagated in cultures of type A strain Hall. The phage DNAs were exclusively synthesized after induction with MC. Alpha 1 and alpha 2 DNAs had sedimentation coefficients of 34.0 and 30.6 S, corresponding to molecular weights of 31.9 x 10(6) and 23.5 x 10(6), respectively. The buoyant density in CsC1 was 1.682 g/cm3 for alpha 1 DNA and 1.680 g/cm3 for alpha 2 DNA. Based on thermal denaturation characteristics, the genomes of both phages were shown to be double-stranded DNAs. Agarose gel electrophoretic profiles of the phage DNAs digested with restriction endonuclease EcoRI revealed nine fragments for alpha 1 DNA and six fragments for alpha 2 DNA. The molecular weights of the phage DNAs as determined by restriction enzyme analysis were 30.55 x 10(6) for alpha 1 DNA and 25.83 x 10(6) for alpha 2 DNA. Nontoxigenic mutants obtained from strain 190L could, like the toxigenic parent strain, produce the two phages after treatment with MC. Lysogenic conversion to toxigenicity by phage alpha 2 was not observed with the nontoxigenic mutants. It seems likely that there is no relationship between either phage genome and the toxigenicity of C. botulinum type A.     

Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica)

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster’s cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

Temperature effects on the viable but non-culturable state of Vibrio vulnificus

Abstract The non-culturable state of Vibrio vulnificus , strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 10⁷ cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 10⁶ cells/ml. Cells at all higher temperatures remained culturable (at least 10⁴/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus , as well as those of six other clinical and environmental strains of V. vulnificus , were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. ( V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus , and V. campbelli ) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.     

Pathogenic potential of a collagenase gene from Aeromonas veronii

The role of collagenase as a mechanism of bacterial pathogenicity in some pathogenic bacteria has been reported. The information on the role of collagenase in Aeromonas spp. pathogenesis is scant. In the present study, a mutant Aeromonas veronii RY001 that is deficient in the putative collagenase gene acg was constructed and compared with the wild-type strain for virulence factors. Bacterial cells and cell-free extracellular products of the mutant had significantly less collagenolytic activity, but there were not significant differences in caseinolytic, gelatinolytic, and elastolytic activities. Adhesion and invasion abilities of the mutant strain on epithelioma papillosum of carp cells was only 56% of that of the wild-type strain, and the cytotoxicity of the mutant strain to epithelioma papillosum of carp cells was only 42% of that of the wild-type strain. The LD50 values of the wild-type strain were determined as 1.6 x 10(6) and 3.5 x 10(5) cfu in goldfish and mice, respectively, whereas the mutant RY001 strain showed slightly higher values (i.e., 2.8 x 10(6) and 1.4 x 10(6) cfu in goldfish and mice, respectively). These results indicated the involvement of the collagenase gene in the pathogenesis of A. veronii.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Mechanism of action of nalidixic acid on Escherichia coli. Vi. Cell-free studies

The effects of nalidixic acid in vitro on deoxyribonucleic acid (DNA)- polymerase (deoxyribonucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), deoxyribonucleotide kinases (ATP: deoxymono- and diphosphate phosphotransferases), and deoxyribosyl transferase (nucleoside: purine deoxyribosyltransferase, EC 2.4.2.6) were examined employing partially purified and crude extracts of Escherichia coli ATCC 11229 and E. coli 15TAU. Nalidixic acid had no inhibitory effect on the DNA-polymerase of the wild-type strain E. coli ATCC 11229 at concentrations of 1.4 x 10(-3) to 2.8 x 10(-3)m. No inhibition of deoxyribonucleotide kinase activity was observed at concentrations of nalidixic acid ranging from 2 x 10(-3) to 8.6 x 10(-3)m. Nalidixic acid (0.43 x 10(-4) to 0.43 x 10(-3)m) had no inhibitory effect on the deoxyribosyl transferase activity of crude extracts obtained from E. coli ATCC 11229 or E. coli 15TAU. Analytical CsCl density gradient centrifugation demonstrated that the DNA obtained after treatment of E. coli 15TAU with nalidixic acid was not cross-linked. These results suggest that the prevention of DNA synthesis in vivo by nalidixic acid is not attributable to inhibition of DNA polymerase, deoxyribonucleotide kinase, deoxyribosyl transferase, or to cross-linking of the DNA of treated cells.     

Self-transferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecalis var. zymogenes.

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Ben-), and those from strain JH3 had also lost resistance to the bacteriocin (Bnr-). The majority of Hly-, Ben- variants from JH1 retained bacteriocin resistance (Bnrplus). Strains JH1 and JH3 contained a plasmid deoxyribonucleic acid species of molecular weight 38 times 10-6 (plasmids pJH2 and pJH3, respectively), and strain JH1 also contained a 50 times 10-6 molecular weight plasmid (pJH1) which has previously been shown to carry the genes determining resistance to the antibiotics kanamycin, neomycin, streptomycin, erythromycin, and tetracycline. Hly-, Bcn-, Bnr- variants of strain JH3 had completely lost plasmid pJH3. Hly-, Bcn-, Bnr- variants of strain JH1 had completely lost plasmid pJH2 and retained plasmid pJH1, but Hly-, Bcn-, Bnrplus variants had retained both plasmids pJH2 and pJH1. The Hlyplus, Bcnplus, Bnrplus traits from both parental strains were transferable to nonhemolytic S. faecalis strains during mixed incubation in broth at 37 C, and hemolytic recipient strains were found to have received plasmid pJH2 from strain JH1 and pJH3 from JH3. We conclude that the Hlyplus, Bnrplus traits are borne on plasmid pJH2 in strain JH1 and pJH3 in strain JH3 and that, in Hly-, Bcn-, Bnrplus variants of strain JH1, plasmic pJH2 has suffered a mutation affecting hemolysin and bacteriocin expression. We infer that the plasmids transfer by conjugation. Beta-hemolytic activity is the only property distinguishing the zymogenes variety from S. faecalis. Since we have shown that this activity is plasmid borne in strains JH1 and JH3, we endorse the view that the varietal status of zymogenes should be dropped.     

Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood

The virulence factors of Vibrio vulnificus are not yet well understood. So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism. The present research was carried out in order to study the presence of some of these enzymes in 133 V. vulnificus strains isolated from 45 seafood samples. The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99·2% for caseinolytic protease, 96·9% for DNase, 65·4% for mucinase and 46·6% for elastase. None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells. In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59·4% of strains showed opaque morphology on BHI agar and 57·9% on nutrient agar, 10·5% presented translucent morphology on both agars and 30·1 and 31·6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively. None of the translucent colonies was virulent to mice. Therefore, opacity was a useful marker for potential virulence. Of 45 food samples contaminated with V. vulnificus , 29 (64·4%) presented strains lethal to adult mice.     

Ultrastructure of muscle cells in the adductor of the boring clam Tridacna crocea

The ultrastructure of the adductor muscle of the boring clam (Tridacna crocea) was investigated. The adductor was composed of opaque and translucent portions. The opaque portion contained smooth muscle cells; the translucent portion contained obliquely striated cells. Smooth muscle cells were classified, according to the statistically analyzed diameters of their thick myofilaments, into two types, S-1 and S-2. S-1 cells had thick myofilaments, 50–60 nm in diameter. S-2 cells had thick myofilaments of two sizes, about 55–65 nm and 85–100 nm in diameter, respectively. Obliquely striated muscle cells in the translucent portion were also classified into two types: O-1 cells, with thick myofilaments 30–35 nm in diameter, and O-2 cells, with myofilaments of 50–60 nm.     

Repair of Pyrimidine Dimer Damage Induced in Yeast by Ultraviolet Light

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.     

Biochemical Studies of Two Bacillus pumilus Plasmids

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm(3) and a molecular weight of about 28 x 10(6). Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm(3) and a molecular weight of about 6 x 10(6). Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.     

Circular deoxyribonucleic acid from Shigella dysenteriae Y6R

Circular deoxyribonucleic acid was isolated from Shigella dysenteriae Y6R and was found to consist of six species having molecular weights of 10(6), 1.3 x 10(6), 2.6 x 10(6), 3.8 x 10(6), 20 x 10(6), and 24 x 10(6) daltons. These size classes were partially resolved by sucrose density gradient centrifugation. The minicircles (10(6) and 1.3 x 10(6)) were found to have a buoyant density in CsCl of 1.710 g/ml. The 3.8 x 10(6) dalton class had a density of 1.707 g/ml. The two largest species had a density of 1.702 g/ml. Two other strains, S. sonnei II and S. dysenteriae 60, also contained circular deoxyribonucleic acid.     

Spirochaeta aurantia, a Pigmented, Facultatively Anaerobic Spirochete

A strain of Spirochaeta aurantia was isolated from mud by a procedure involving migration of the organisms through cellulose ester filter discs (0.3-mum pore diameter) onto the surface of culture plates. The helical cells measured 0.3 by 10 to 20 mum during exponential growth. Electron microscopy showed the presence of two subterminally inserted axial fibrils partially overlapping in a 1-2-1 arrangement. An outer envelope, exhibiting a polygonal substructure, was observed. The spirochete grew either aerobically or anaerobically, with aerobic yields of 9.8 x 10(8) cells per ml and anaerobic yields of 3.0 x 10(8) cells per ml. The organism used carbohydrates, but not amino acids, as energy sources. Amino acids served as sole nitrogen sources, whereas inorganic ammonium salts did not. The presence of biotin and thiamine in the medium was required for growth. Growing cells fermented maltose mainly to carbon dioxide, hydrogen, ethyl alcohol, and acetic acid. Small amounts of formic and lactic acids, acetoin, and diacetyl were produced. Cells of S. aurantia growing aerobically produced a yellow-orange pigment. Chemical analysis indicated that the pigment was carotenoid in nature, its main component being lycopene or a similar compound. S. aurantia is not closely related to the leptospires, since it lacks both the hemolytic antigen and the hooked ends typical of the latter organisms. Furthermore, the guanine plus cytosine content in the deoxyribonucleic acid of S. aurantia (66.8 moles%) differs drastically from that of leptospires.