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Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.

Vibrionaceae

R plasmids in vibrionaceae - beta-lactamases in Vibrio cholerae (NAG-Heiberg II) and A. hydrophyla.

290 natural isolates of Aeromonas, Plesiomonas and Vibrio strains isolated from surface waters have been tested for antibiotic resistance, and 123 have been found resistant to various drugs, mostly beta-lactam antibiotics. 14 of them, with high-level of resistances, transfered antibiotic resistance using three recipient strains of Enterobacteriaceae. One strain of Vibrio NAG (Heiberg II) transfered the resistance to ampicillin, carbenicillin and cephaloridin to both E. coli K12 3110 and S. typhimurium LT2 recipient, and further, in the second cycle of transfer, to E. coli K12 185 Nx strain. One additional strain of Aeromonas hydrophyla transfered a high-level ampicillin- and a low-level gentamicin resistance to E. coli K12 3110 only. Here, the resistances could not be transfered in the second cycle, and, they did not occur in the same exconjugant colony. This confirms previous reports that antibiotic resistance is widely distributed among strains found in wild nature.

Aeromonas

Distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species.

The distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species was examined. Enzyme activity was demonstrated in cell-free growth media from all members of the family Vibrionaceae which were tested except Plesiomonas shigelloides. In each case, enzyme was produced in exponential to early stationary phase and was excluded from Sepharose 6B, indicating a complex of high molecular weight. In a limited survey of other families, Stahylococcus aureus was the only organism outside the Vibrionaceae which was shown to produce the enzyme. In this case, however, the enzyme exhibited much less activity against erythrocyte membranes and appeared to have a lower molecular weight. The reasons for these differences and the importance of the acyltransferase as a biochemical identification tool are discussed.

Acyltransferases

Rapid diagnosis of Enterobacteriaceae. I. Detection of bacterial glycosidases.

The paper describes a number of tests for the rapid detection of glycosidases including alpha-glucosidase, beta-glucosidase, beta-glucuronidase, beta-xylosidase and alpha-fucosidase. The methods use heavy suspensions of viable but non-multiplying bacteria in a buffered solution of a chromogenic substrate. The results of the tests are readable within 4 h. The application of these tests to a collection of 633 strains of Enterobacteriaceae and Vibrionaceae demonstrates that some of the tests may be valuable additions to the present tests available for the identification of bacteria belonging to these families. beta-glucuronidase activity was observed only in strains of the Escherichia-Shigella group. 97 per cent of the Escherichia strains possessed beta-glucuronidase activity. beta-xylosidase activity was almost completely restricted to strains of the Klebsiella-Enterobacter group in addition to Yersinia strains. None of the strains possessed alpha-fucosidase activity.

Bacteriological Techniques

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.

Animals

[Similarity of the DNA mucleotide sequences of vibrios].

The systematic position of some Vibrio species was ascertained by the method of molecular DNA -- DNA hybridization. The DNA of the brine vibrios V. costicola and V. fischeri were shown to have about 10% of sequences homologous with DNA of a typical cholera vibrio (V. cholerae eltor No. 334). Similarity between the genomes of other representatives of the Vibrionaceae family, as well as in DNA hybridization of V. costicola and V. fischeri, was found to be approximately on the same level. All species included into the genus, Vibrio on account of their phenotypic characteristics may be considered to have essential differences in the structures of their genomes.

Base Sequence

Rapid identification of Enterobacteriaceae. II. Use of a beta-glucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples.

beta-glucuronidase activity is an exclusive characteristic of E. Coli and some shigellae among Enterobacteriaceae and Vibrionaceae. An agar medium (PGUA agar) which permits the detection of bacteria with beta-glucuronidase activity in mixed cultures was evaluated as a primary culture medium for clinical samples of urine. The medium was selective for enterobacteria and yielded significantly higher recoveries than MacConkey agar. Based on the examination of 3,460 urine samples, it was found that the use of the PGUA agar has several advantages over conventional methods: 1) 94% of all E. coli cultures could be identified on the basis of their appearance on the primary plates; 2) The use of the PGUA method did not result in any misidentidications as compared to 1% of cultured misidentified by the conventional procedure; 3) Approximately one-half of the urine samples which contained E. coli as the sole organism could be reported following the reading of primary culture plates; 4) The application of the PGUA medium resulted in a 46% reduction in the cost of media employed and a 67% reduction in the time required for the processing of urine samples.

Agar

Molar growth yields, respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a chemostat.

The effect of growth rate on the physiology of Beneckea natriegens was studied in chemostat culture. The molar growth yields (Y) from glucose and oxygen, the specific rates of oxygen (Qo2) and glucose (Qg1c) consumption and the specific rate of CO2 production (Qco2) were linearly dependent on the growth rate over the dilution rate 0.17 h-1 to 0.60 h-1. Further increase in the dilution rate resulted in a decrease in growth yield and respiration rate and these changes were coincident with increases in the specific rate of glucose utilisation and of acetate production. The affinity of Beneckea natriegens for glucose was similar when measured either directly in chemostat culture or in a closed oxygen electrode system using harvested bacteria. The total content of cytochromes decreased with increasing growth rate. However, the quantity of CO-binding cytochromes remained independent of growth rate and correlated with the potential respiration rate.

Carbon

An ultraviolet light induced bacteriophage in Beneckea gazogenes.

An ultraviolet light induced prophage has been discovered in the red pigmented marine vibrio Beneckea gazogenes. Two spontaneously derived pigment mutants, one forming pink colonies and one lacking pigment and forming white colonies, were also irradiated. The presence of pigment was not related to phage induction; uv-induced cell lysis occurred in wildtype and mutant strains at the same dosages. Lysis was not prevented or retarded by exposure after irradiation to visible light indicating the phenomenon was not photoreactivable. Electron micrographs of the 'T-like' B. gazogenes phage are shown. A second beneckea was isolated form the anaerobic zone of cyanobacterial mats growing in the hypersaline environment of Laguna Mormona, Baja California. The Baja beneckea does not harbor a uv inducible prophage and is resistant to the B. gazogenes phage under all conditions tested.

Bacteriolysis

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.

Flavin Mononucleotide

Activity and stability of the luciferase--flavin intermediate.

A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.

Anaerobiosis