Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

Taxonomic studies on some gram-positive coryneform hydrogen bacteria

Recently isolated coryneform hydrogen bacteria were investigated under taxonomical aspects. Strains 7 C, RH 10, and 14 g are characterized by the snapping type of cell division, 68.5 to 69.7% GC content, dl-diaminopimelic acid in the cell wall, content of metachromatic granules, weak utilization of sugars and inhibitory effect of citrate. The strains are placed to the group 1—genus Corynebacterium—of the classification of coryneform bacteria of Yamada and Komagata (1972) and the name Corynebacterium autotrophicum sp.nov. is proposed.Strains 11 X and RH 12 are characterized by the bending type of cell division, a GC content of 70.2 and 70.5%, ll-diaminopimelic acid in the cell wall, absence of metachromatic granules, utilization of several sugars and no changes in cell morphology by citrate. The strains have to be placed to group 6 of coryneform bacteria.     

Bioaccumulation of silver by a multispecies community of bacteria

A stable community of bacteria that had unusually high tolerance of soluble silver was isolated from soil by chemostat enrichment. The community consisted of three bacteria: Pseudomonas maltophilia, Staphylococcus aureus and a coryneform organism. The pseudomonas was primarly responsible for the silver resistance. The tolerance of high silver concentrations, up to 100 mM Ag⁺, was greatly reduced when the community was grown in the absence of silver. Pseudomonas maltophilia comprised approximately 50% by numbers of the community when grown in chemostats in the presence or absence of Ag⁺ but large fluctuations occurred in population sizes of the other two bacteria; the S. aureus population was small (less than 1%) in the presence of Ag⁺ but comparised a third of the total numbers when Ag⁺ was omitted from the medium. Silver-resistant respiration of the silveradapted community was significant even when it was confronted with high concentrations of Ag⁺. In contrast the respiration of the coryneform organism and particularly S. aureus was highly sensitive to silver. The inhibition constants for silver-sensitive respiration were 0.78 mM and 0.04 mM for silver acclimatized and nonacclimatized communities respectively.The community had great capacity for silver bioaccumulation. Maximum concentrations of over 300 mg silver per g dry weight of biomass were recorded at an accumulation rate of 21 mg Ag⁺ h^-1 (g biomass)^-1. The extent of silver removal from solution was a function of initial concentration of silver; at low external concentrations (ca. 1 mM) all the silver was rapidly removed from solution, at high concentrations (ca. 12 mM) 84% removal occurred in 15 h.     

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.     

Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg-matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.     

Immunofluorescent Localization of Z-Nin in the Z-Disk

Of 14 coryneform and 2 Micrococcus strains tested, Arthrobacter globiformis IFO 12137, A. simplex IFO 12069, and Brevibacterium helvolum IFO 12073 utilized l-arginine as a sole carbon and nitrogen source, and synthesized the enzymes specific for the arginine oxygenase pathway when grown on l-arginine. The first step reaction was stimulated by FAD and aeration, and the enzyme responsible was shown to be arginine 2-monooxygenase (EC 1.13.12.1). High activities of five enzymes, including guanidinobutyramidase and ganidinobutyrase (EC 3.5.3.7), were detected in the extract of l-arginine-grown A. simplex cells. The enzymes in the last two steps, 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase (EC 1.2.1.16), of B. helvolum were also induced by putrescine. These results indicate that some bacteria belonging to the coryneform group employ the arginine oxygenase pathway as a major route for l-arginine metabolism, l-arginine being degraded to succinate via 4-guanidinobutyramide and 4-guanidinobutyrate. The last part of the pathway may be common to the pathway for putrescine degradation.     

Capacity of siderophore — producing alkalophilic bacteria to accumulate iron,gallium and aluminum

Summary The ability of alkalophilic bacteria to remove iron, gallium and aluminium from culture media is reported. Of six bacterial strains grown in the presence of iron, gallium or aluminium (10 M), five were able to accumulate iron or gallium, but only two depleted the aluminium stock. A comparison of gallium removal under low (< 1 gmM) or high (10 gmM) iron conditions showed that two isolates accumulated gallium only under low-Fe conditions. One isolate, a coryneform bacterium, was able to grow in the presence of 1, 10 and 100 mg gallium/l, but growth and siderophore production were affected at high gallium concentration. Similar concentrations of gallium were accumulated from cultures initially containing 1 or 100 mg gallium/l. Offsprint requests to: D. J. Gascoyne     

Transposon mutagenesis of coryneform bacteria

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10⁴ mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.     

N2-Fixation by chemoautotrophic hydrogen bacteria

The Gram-positive coryneform bacteria strains 14g and 7C were found to be able to grow with N₂ as sole nitrogen source when incubated under microaerobic conditions. Nitrogenase activity in whole cells was assayed by acetylene reduction. High rates of ethylene production (50–120 nmole/hxmg cell protein) were observed in N₂ or glutamate grown cell suspensions shaken in an atmosphere of 2.5% O₂, 10% acetylene and 87.5% argon.     

Numerical Classification of Bacteria

Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods—Sneath’s and Lockhart’s systems—, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups, especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless of which coding method was applied.

Though animal corynebacteria have conventionally been considered to occupy the taxonomic position neighboring to genera Arthrobacter and Cellulomonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.     

Brevibacterium sp. from poultry

Two isolates of methanethiol producing coryneform bacteria sharing morphological and physiological similarities with Brevibacterium are described. They were isolated from bumble-foot-like manifestations of poultry but proved to be non-pathogenic for experimental animals.     

The production of auxins by the endophytic bacteria of winter rye

The ability of the actinomycetes and coryneform bacteria isolated from the root tissues of winter rye to produce auxin in a liquid culture was studied. The isolates of coryneform bacteria produced indolyl-3-acetic acid (IAA) into the medium in the amount of 9.0–95.0 μg/ml and the isolates of actinomycetes in the amount of 39.5–83.0 μg/ml. The maximal IAA accumulation in culture liquid of actinomycetes coincided, in general, with the beginning of the stationary growth phase. The dependences of IAA synthesis by actino-mycetes on the composition and pH of nutrient medium, tryptophan concentration, and aeration conditions were determined. Biological activity of the bacterial IAA was assessed. Treatment of winter rye seeds with coryneform auxin-producing bacteria increased the germination capacity and enhanced an intensive seedling growth in vitro.     

Glutamicin CBII,a bacteriocin-like substance produced by

Corynebacterium glutamicum CBII, in the stationary phase of growth, was found to produce spontaneously a substance resembling bacteriocins by its bactericidal properties. This substance designated glutamicin CBII was observed to exhibit bactericidal activity against coryneform bacteria (12 species tested) but not against unrelated gram-positive (3) and gram-negative (3) bacteria, while its action on bacteria with no quite known relatedness to the coryneform group (14) was found to be variable. Glutamicin CBII was partially purified by precipitation with ammonium sulphate (70% saturation), selective heat precipitation and gel chromatography on Sepadex G-50. The antibacterial substance diffused through cellophane membrane with an approximate cut-off of 10000 dalton and its sedimentation coefficient was determined to be 1.1. S by ultracentrifugation. Heating at 100°C for 30 min had no effect on its activity. Glutamicin CBII was proved to be resistant to chloroform, trypsin, chymotrypsin, pronase, and subtilisin. According to its staining behaviour and ¹H NMR spectra it probably represents a glycoprotein containing only a minor protein component.

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Enrichment and isolation of nitrogen fixing hydrogen bacteria

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by ¹⁵N₂ incorporation.     

Adherence of Yersinia enterocolitica to Mammalian Epithelial Cell Lines

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y-1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature-dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A-methyl-A-nitro-A-nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C. When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells. Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria. These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature-dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.     

Comparative study of soil bacterial flora as influenced by the application of a pesticide,pentachlorophenol (PCP)

Summary The effects of pentachlorophenol (PCP) applications on the taxonomic composition of bacterial microflora were studied in water-logged soil (WS) and in shake cultures of suspended soil (SS). PCP applications resulted in a predominancy of Gram-negative bacteria over Gram-positive species. Members of theAcinetobacter group were the most common in PCP-treated soil although a small portion of the flora were in thePseudomonas-Alcaligenes group or belonged to theEnterobacteriaceae. Coryneform bacteria and species of theBacillus were the dominant forms in untreated WS; however, WS cultures treated with PCP at recommended rates (2.67 gm/m²) evidenced species ofPseudomonas, Alcaligenes, Acinetobacter, and members of theEnterobacteriaceae as the predominant bacterial species. The dominance of Gram-negative bacteria in PCP-treated soil was evidenced for 3 months after application of the compound but was not evident after 17 months when PCP had dissipated. Gram-negative bacteria found in PCP-treated soil were highly tolerant of the phenol. In WS cultures coryneform bacteria were the most common although PCP tolerance was heterogenous in nature.     

Identification of Rhodococcus fscians (Tilford 1936) Goodfellow 1984

Determinative laboratory tests which distinguish Rhodococcus fasdans from all other coryneform plant pathogenic bacteria were used to compare strains of this species with representative strains of Clavibacter spp. and Curtobacterium spp. The strains were also compared by colony hybridization using genomic DNA from R. fascians as a probe. Only strains of R. fascians authenticated with the determinative tests gave positive reactions to the probe. The pathogenicity of strains was tested in seedlings of Lathyrus odoratus L. (sweetpea) grown in soil, and asepticaily in agar in tubes. Fasciation was induced in sweetpea by 20 strains of R. fascisms from Begonia sp., Brodiaea laxa, Carica X heilbornii nm. pentagona, C. pubescens, Dahlia sp., Fragaria X ananassa, Lathyrus odoratus, and Verbascum nigrum. Strains of R. fasdans from Chrysanthemum sp., Gladiolus sp., and Justicia brandegeana did not induce fasciation in sweetpea.     

Endophytic bacteria in sunflower (<Emphasis Type="Italic">Helianthus annuus </Emphasis>L.): isolation,characterization, and production of jasmonates and abscisic acid in culture medium

This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Further Characterization of Bacterial Production of Anthranilic Acid from Aniline

The production of anthranilic acid (AnA) was investigated for 40 bacterial strains in the presence and absence of aniline. Resting cells of all aniline-assimilating bacteria tested produced AnA with aniline, but not without aniline. The cells of aniline-assimilating Rhodococcus erythropolis strains produced more AnA than those of other aniline-assimilating bacteria. Resting cells of several non-aniline-assimilating strains produced AnA in the absence of aniline. However, its production by these strains was much lower than that by the Rhodococcus strains. The production of AnA by cells of aniline-assimilating R. erythropolis AN-13 was promoted by aliphatic monocarboxylates, ATP, biotin and coenzyme A, and repressed by catechol analogues, N-ethylmaleimide and iodoacetate. On the other hand, its production by non-aniline-assimilating Pseudomonas sp. AN-21 was repressed by glucose, mannose and some amino acids.