Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322 bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.     

Cadmium resistant Enterobacter cloacae and Klebsiella sp. isolated from industrial effluents and their possible role in cadmium detoxification

Three bacterial strains, two of Klebsiella sp. and one Enterobacter cloacae were isolated from industrial effluents of chemical and textile industries. They showed high efficiency in removing cadmium (Cd²⁺) from the medium. When 100 g/ml of Cd was added to the medium, the three isolates namely CMBL-Cd1, CMBL-Cd2 and CMBL-Cd3 removed or accumulated 86%, 87% and 85% of Cd, respectively, from the medium within 24h. Plasmids were detected in all the three strains. Plasmids of E. cloacae (pCBL1) and Klebsiella sp. (pCBL2 and pCBL3), estimated to be 6.6kb, were used to transform Escherichia coli C600. The transformed E. coli cells showed elevated resistance to Cd. Ethidium bromide curing indicated the presence of the Cd resistance gene on the plasmid. Resistance of the isolated strains against other metals like chromium (cr⁶⁺) and lead (pb²⁺) and a number of antibiotics was also checked. Cured strains showed lowered resistance against Cr and some antibiotics. This again supported the indication of the presence of Cd, Cr and some antibiotics resistance genes on plasmids.     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Plasmid instability inLactobacillus plantarum strain caTC2

The stability of plasmids inLactobacillus plantarum was investigated by extended incubation of bacterial cells in the presence of different carbohydrates. Strain caTC2, carrying a plasmid-encoded chloramphenicol-resistant (Cm^r) phenotype, was grown overnight (16–18 h) in MRS, MRS-L, and MRS-M broths containing 2% glucose, lactose, and maltose respectively at 30°C. The cultures were subsequently held at 30°C and room temperature (21±1°C) for an extended period (7 days). The total viable cell counts were assayed on MRS agar plates and tested for sensitivity to 30 g chloramphenicol/ml by replica plating. The plasmid profiles of the chloramphenicol-sensitive strains showed that there was a loss of the 8.5-kb plasmid, but not the 10.6 or 6.5 kb plasmids. Concomitant loss of the chloramphenicol resistance phenotype and plasmid at high frequency, particularly by using MRS-L at 21°C method, suggests that this would be a simple and efficient method for curing selected plasmids in lactobacilli.Contribution No. 2039 from the Centre for Food and Animal Research.     

Genetic transformation of the pathogenic fungus Wangiella dermatitidis

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10–203 HmB-resistant colonies/g pAN7-1 on medium containing 100 g HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20–40 g/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17–50 transformants/g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.     

Plasmid incidence in bacteria from agricultural and industrial soils

Heavy metal contents of agricultural and industrial soils were determined by atomic absorption spectrophotometry. The analysis of the samples collected from two different locations revealed significantly high levels of Fe, Zn, Cu, Cr and Ni. Certain microbiological parameters (total aerobic heterotrophs, asymbiotic N₂-fixers, total Actinomycetes and fungi) were also monitored from these soils. A total of 70 bacterial isolates from agricultural and industrial soils were examined for plasmid DNA content and resistance to the antibiotics amoxycillin, cloxacillin, chloramphenicol, doxycycline methicillin, nalidixic acid, and tetracycline. Minimum inhibitory concentrations (MICs) of Cu, Cr, Pb, Cd, Hg, Zn, and Ni for each isolate were also determined. Resistance was most frequent to methicillin (48.5%), cloxacillin (45.7%), and nalidixic acid (40%) for all isolates of bacteria. The highest MICs observed were 100 g/ml for mercury, 800 g/ml for Ni and 1600 g/ml for other metals. The incidences of metal resistance and MICs of metals for bacteria from industrial soil were significantly different to those of agricultural soil. On a percentage basis, 91.4% of the total bacterial isolates from industrial soil were found to harbour plasmids whereas 40% of the isolates from agricultural soil contained plasmids.     

Filterable marine bacteria found in the deep sea: Distribution,taxonomy, and response to starvation

A significant number of viable colony-forming bacteria were recovered from deep-ocean bottom water samples passed through a 0.45m filter. However, these bacteria small enough to pass through a 0.45m membrane filter and termed filterable bacteria were less abundant in open-ocean surface water and coastal water samples. The reduced size of bacterial cells present in deep-ocean bottom water samples was documented by scanning electron microscopy. The concentration of ATP in the water samples was found to be correlated with results of direct counts of bacteria.Numerical taxonomy of bacterial strains isolated from water samples collected at two stations in the deep sea yielded taxonomic clusters grouped according to sample and size fraction. The generic composition of bacterial populations of bottom water filtrates was compared with that of bacteria retained by 0.45 m filters. Strains ofAlcaligenes, Flavobacterium, Pseudomonas, andVibrio spp. were identified among those retained by, as well as passing through, 0.45m filters.Two marine isolates obtained from the filtrate of a deep-ocean water sample were incubated for 9 weeks in nutrient-free artificial seawater, during which the cells became rounded and reduced in size. After the 9-week incubation period, more than 10% of the viable cells of both cultures were able to pass through a 0.4m filter. The viable count at 9 weeks wasca. 10% of that of the initial population, although from direct counts the total population number remained relatively constant throughout the incubation period. From the observed reduction in cell size and increased starvation resistance of cells held under low nutrient conditions, it is concluded that a significant relationship exists between decreased cell size and increased survival of marine bacteria in the deep sea.This investigation was supported by Grant No. OCE 76-82655 from the National Science Foundation.     

Transformation of commercial baker's yeast strains by electroporation

Summary We developed an electroporation protocol for transformation which was particularly optimized for commercial baker's yeast strains. The protocol is based on the standard BIORAD GENE PULSER/PULSE CONTROLLER machine. It works efficiently both for the introduction of standard multicopy plasmids (ARS and 2m based) and for integrative transformation. In particular we were able to transform genuine prototrophic baker's yeast strains with a 2m-based multicopy plasmid, carrying the dominant sulfometuron methyl resistance marker. For plasmids requiring the introduction of more than one copy for complementation, the transformation frequency was considerably lower. This suggests that transformation by the electroporation method introduces on average only one or a few copies of the transforming plasmid per cell.     

A study of the organisation of the ribosomal ribonucleic acid gene cluster of Neurospora crassa by means of restriction endonuclease analysis and cloning in bacteriophage lambda.

Summary Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 m deletion and pAP2 by two deletions — the same 8 m deletion and a further 9 m deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 m, a buoyant density of 1.715 g/ml and a copy number up to 65.     

Cryptic plasmid pBf1 fromButyrivibrio fibrisolvens AR10: Its use as a replicon for recombinant plasmids

A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacteriumButyrivibrio fibrisolvens strain AR10, ampped with restriction endonucleases, and cleavage sites suitable for attachment toEscherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42°C or in 0.25 g ampicillin/ml, but growth in 50 g acridine orange/ml for three culture passages produced cured colonies at a frequency of <1%. Chimeric plasmids were constructed by combining pBf1 with theE. coli plasmid pUC18, in addition to the clindamycin resistance gene fromBacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene fromE. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.     

Polymorphism of 2-μm plasmids in industrial strains of Saccharomyces cerevisiae

Restriction fragment length polymorphism (RFLP) analyses of industrial Saccharomyces yeast DNA have identified eight 2-m plasmidsvariants that fall into two distinct types. Type-I plasmids are of unique form, whereas type-II plasmids exist in seven distinct RFLP forms. Only two different 2-m variants were observed in 35 bakers' strains analysed. One variant was the unique type-I whereas the second variant represents an ancestral form of the type-II plasmid. Sixteen of nineteen wine yeasts carried a distinctive type-II plasmid with a homeologous STB repeat whereas ale and lager yeasts had a wide range of type-II variants. Relative to nuclear and mtDNA, 2-m polymorphism is less diverse and not diagnostic for a specific strain. This 2-m DNA polymorphism is a convenient and useful addendum to nuclear and mtDNA RFLP analyses but cannot serve as the sole marker for strain identification. A tentative phylogeny of industrial S. cerevisiae yeasts is suggested with origins in bakers' yeast carrying the ancestral type-II form. Correspondence to: G. H. Rank     

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentK _m values of 1.1 M and 10 M for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Plasmids in Nitrobacter

A screening of nine Nitrobacter strains showed that Nitrobacter X14 and Nitrobacter Y possess plasmids designated as pNH1 and pNH2, respectively. The plasmids pNH1 and pNH2 had molecular weights of about 76 Mdal as estimated by agarose gel electrophoresis. They showed similar cleavage patterns when digested with EcoRI, HindIII or BamHI. Electron microscopic investigations exhibited that the plasmid pNH1 had a contour length of 36.9 m corresponding to a molecular weight of 76 Mdal.     

Characterization of chloramphenicol resistance inLactobacillus plantarum caTC2

A strain ofLactobacillus plantarum caTC2R isolated from a meat source was resistant to chloramphenicol (30 g/ml). Resistance was mediated through an inducible chloramphenicol acetyltransferase. Plasmid analysis of this strain showed three plasmids, of which the 8.5-kb plasmid apparently encodes the gene for chloramphenicol resistance. This plasmid was lost at high frequency (25%) when theLactobacillus was subcultured at a higher than optimal temperature (40°C).     

Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient.

Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 m yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 m DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 m-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura⁺ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5 monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.These features should offer new possibilities for cloning with yeast.     

Respiratory studies on two small freshwater amoebae

The changes in respiration rate and mean cell volume induced by temperature within the range 10°C–25°C were investigated in two small species of freshwater amoebae,Saccamoeba limax Page andVannella sp. Mean cell volume varied in response to temperature, with maxima at 20°C inVannella sp. (10.15× 10³ (±1.80)m³ and 15°C inS. limax (9.08×10³ (±0.93)m³. Respiration rate increased over the temperature range investigated. The highest rates and the greatest rate of increase between temperatures occurred inVannella sp. Q₁₀ ranged between 0.12 and 1.33 inS. limax and between 1.77 and 7.36 inVannella sp. A regression of log oxygen uptake versus log cell volume incorporating the data of the present investigation and the data of other workers on amoeba respiration is presented, and the ecological significance and application of such data discussed.     

Micropropagation of Phytolacca dodecandra through shoot-tip and nodal cultures

A procedure for micropropagation of endod (Phytolacca dodecandra) is described. BA at 0.44 M produced 3.1 new shoots per expiant in six weeks using shoot tips. Nodal expiants, however, produced up to 4.7 shoots per explant on medium with 0.44 M BA and 0.27 M GA,. IBA at 0.49 M induced 90% rooting with minimal callus. Plantlets were successfully transferred to the greenhouse and some staminate clones produced flowers after six months.Abbreviations BA 6 benzylaminopurine - kinetin 6-furfurylaminopurine - 2iP N⁶-(2-isopentyl)adenine - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - GA₃ Gibberellic acid - IBA indole-3-butyric acid     

Tributyltin-resistant marine bacteria: a summary of recent work

Summary A tributyltin chloride (TBTCl)-resistant bacterium,Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 M TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by whichE. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment fromHindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage andE. coli. TBTCl-tolerant marine bacteria other thanAlteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments fromAlteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.