Variability of endonucleolytic activity indicates high genetic diversity within the natural population ofSelenomonas ruminantium

The population of bacteria ofSelenomonas ruminantium species in the rumen of fallow-deer was analyzed using endonucleolytic activity assay and plasmid profiles. This analysis indicated a high diversity within the population ofS. ruminantium. At least 12 different restriction profiles, indicating the presence of the different specificity nucleases, have been observed. Site-specific endonucleases were detected in 17 out of 45 strains tested. In other strains a various level of nonspecific activity was detected. Plasmid DNAs ranging in size from 0.9 to more than 25 kbp were detected in 60% of strains analyzed. No or little correlation was observed between the endonuclease activity and the plasmid content. The presence of different specificity endonucleases, as well as differences of plasmid profiles of isolates possessing identical specific activity indicate that the population ofS. ruminantium in the rumen of an individual animal consists of at least 10 different clones. This work was supported byGrant Agency VEGA 3007/96.     

Characterization of a cryptic plasmid (p0M1) inButyrivibrio fibrisolvens by restriction endonuclease analysis and its cloning inEscherichia coli

A small cryptic plasmid has been identified in a strain of the ruminal bacteriumButyrivibrio fibrisolvens. This plasmid has been isolated and purified. It is approximately 2.8 kbp in length and contains restriction sites for a number of common endonucleases including single sites for EcoRI, PvuII, and PstI. A map of the plasmid restriction sites has been constructed. This plasmid, designated p0M1, has been ligated to pBR325, pAT153, and pHV33 and transformed intoEscherichia coli, and the resulting hybrid plasmids have been mapped. The possible uses of such hybrid plasmids for gene cloning inB. fibrisolvens are discussed.     

Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

Occurrence of pS86/pEF47-Related Plasmids in Gram-Positive Cocci

A small, low-copy-number cryptic 5.5-kb plasmid, designated pEF47, was isolated from a rumen bacterium Enterococcus faecalis 47 and partially characterized. Comparisons by restriction and partial sequence analysis of pEF47 demonstrated high homology to pS86 plasmid, recently isolated from a human clinical strain E. faecalis S-86. PCR followed by sequence analysis and Southern hybridization showed that pS86/pEF47-related replicons are regularly encountered in plasmids from Gram-positive cocci.     

Stimulation of bacterial DNA transformation by cattle saliva: implications for using genetically modified plants in animal feed

To investigate the likelihood of DNA transfer from genetically modified plants (GMP) to bacteria, a rescue plasmid system for Streptococcus gordonii was modified. It was applied to monitor the DNA transformation into oral and intestinal bacteria in cattle. Transformation and recombination frequency of S. gordonii was dependent on the length of the transformed DNA. Beside horse serum, cow saliva also rendered the cells competent for DNA uptake. Competence induction was completely abolished by the addition of liquid from maize silage. Competence was partially suppressed by the addition of rumen liquid. In order to study native bacteria, 724 colonies sensitive to the antibiotics were isolated from either silage or the saliva and rumen of cows. Using horse serum, silage liquid, cow saliva or rumen liquid for competence induction, the isolates failed to integrate linearized pMK110 DNA and restore antibiotic resistance. Only 6 of the colonies obtained from the teeth of a silage-fed cow were sensitive to the antibiotics. Two isolates were related to Staphylococcus warneri. They could be transformed with the model plasmid pMK110 after induction by horse serum. DNA transformation, however, was not stimulated by incubation with cattle saliva, silage or rumen liquid. The response to competence-stimulating factors seems to vary between different bacterial species. These results suggest that the probability of DNA uptake from silage of GMPs is very low.     

Cloning of the integration and attachment regions of bacteriophage P4

Summary The integration and attachment regions of bacteriophage P4 have been cloned into a multicopy plasmid. This plasmid can integrate into the E. coli chromosome at the same location as the parent phage. Integration increases the stability of the plasmid and allows it to be retained even under conditions in which a non-integrated plasmid would be lost. None of the genes needed for P4 lytic growth is required for integration. The P4 integration and attachment regions have been cloned on separate plasmids. A plasmid that carries the attachment site can integrate into the chromosome only if another plasmid that carries the P4 integration functions is present. A plasmid that carries only this trans-acting integration function cannot integrate. Using deletion mutants of the plasmid, the maximum size of the region needed for integration has been determined to be 1.6 kb, of which no more than 1.2 kb codes for the integrase protein. A nonsense mutant defective in integration has been isolated by using a rapid screening procedure that identifies unstable plasmids.     

Genetic diversity in ruminal isolates ofSelenomonas ruminantium

Diversity in the ruminal bacterial speciesSelenomonas ruminantium has been investigated by DNA fingerprinting, DNA-DNA hybridization, plasmid analysis, bacteriophage sensitivity, and monoclonal antibody-based immunoassay. Twenty different isolates from the sheep rumen were initially classified morphologically and by carbon source utilization. DNA fingerprint analyses and quantitative genomic DNA hybridizations showed that limited grouping of these isolates was possible, with the largest group comprising four isolates, and two other groups comprising two isolates each. The remaining isolates were unique. Plasmids in four different size classes, 2.5, 3.7, 6.5 and 12.0 kbp, were identified, but these did not appear in all isolates. There was no apparent relationship between DNA fingerprint pattern and plasmid content. Only three isolates were sensitive to theS. ruminantium-specific temperate bacteriophage S-1. These data indicate that substantial genetic diversity exists within the ruminal speciesS. ruminantium, but that at least one strain may represent up to 20% of isolates.     

Multiresistant strains ofEscherichia coli isolated from the rumen of young calves

Five multiresistant strains ofEscherichia coli were isolated from the rumen fluid of young calves. Resistance to tetracycline and ampicillin was found to be associated with the transfer of a 6.4 kbp plasmid present in two investigated strains.     

Genetic characterization of pAsa6, a new plasmid from Aeromonas salmonicida subsp. salmonicida that encodes a type III effector protein AopH homolog

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536 bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.     

Genetic map of the crown gall suppressive IncW plasmid pSa

Summary A genetic map of the W incompatibility group plasmid pSa has been prepared through the construction of deletion derivatives of pSa and the cloning of various fragments of pSa in pBR322. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to chloramphenicol, sulfonamides, spectinomycin, streptomycin, kanamycin, gentamycin, and tobramycin. Information sufficient for the replication of the plasmid in both Escherichia coli and Agrobacterium tumefaciens is contained within a 4 kilobase pair region. Two regions have been identified as involved in the transfer of the plasmid; one of these regions is also involved in the inhibition of oncogenesis by pSa when it is present in an oncogenic strain of A. tumefaciens. Certain of the deletion derivatives of pSa are potential vectors for the cloning and analysis of A. tumefaciens Ti plasmid DNA.     

Bifid bacteria in bovine rumen

Summary About 500 bifid isolates from 150 samples of bovine rumen liquor were examined for their morphology, physiology and biochemistry. Diagnosis as bifid bacteria was based upon the peculiar pathway of glucose anaerobic metabolism i.e. the fructose-6-phosphate shunt. Four phenetic types were recognized. These types can be differentiated from those found in human habitats because their cell-free extracts are aldolase and HMP dehydrogenases positive: they are potential heterofermenters; furthermore the rumen types are nutritionally different. The distinction of the rumen bifids from the Bifidobacterium species of the intestine of Apis mellifica and Apis indica is still more consistent for a lot of characters. The characters of two rumen types warranted the creation of two new species of the genus Bifidobacterium. One of these, B. globosum n. sp., has a proper morphology, is serologically distinct and has a deoxyribonucleic acid base composition, in % GC, of 64.5. The other, B. ruminale n. sp., found so far only in rumen, is characteristically lactose non fermenter, at variance with all the bifids from human habitats and has peculiar morphological traits. A third type is probably a rough variant of B. ruminale and a fourth is serologically distinct and mannitol fermenter; their taxonomic definition is still, however, premature.This investigation was supported by a grant received from Consiglio Nazionale delle Ricerche (C.N.R.), Roma.     

Isolation and physical characterization of plasmid pCCl from Corynebacterium callunae and construction of hybrid derivatives

Summary The corynebacteria seem to be the most suitable microorganisms for cloning genes involved in the production of amino acids, nucleotides, and other products of industrial interest. A plasmid, pCCl, from Corynebacterium callunae has been found with a size of 4.3 kb. A physical map obtained with restriction endonucleases is presented. pCCl has single restriction sites for Kpn I, Sma I, Bal I, and Hind III. Copy number of this plasmid has been estimated to be about 30. A number of hybrid plasmids have been constructed between pCCl and pBR329 from Escherichia coli and transformed into corynebacteria. The thiostrepton resistance gene (tsr) from Streptomyces azureus has been inserted into them.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes. Received: 30 July 1998 / Accepted: 2 November 1998     

Lectins as markers of rumen epithelial cell differentiation

Summary Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B₄ (Bandeiraea simplicifolia, isolectin B₄)) were explored for use as differentiation markers of rumen epithelial cellsin vivo andin vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA),N-acetylglucosamine (WGA) or forN-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B₄ and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.     

The DNa-protein relaxation complex of the plasmid RK2: Location of the site-specific nick in the region of the proposed origin of transfer

Summary The broad host range plasmid, RK2, has been isolated as a DNA-protein relaxation complex. Nicking of the plasmid DNA in the relaxation complex occurs at a single specific site (rlx) located approximately 20 kb away from the origin of DNA replication. A cis-acting function required for plasmid transfer, the presumptive origin of transfer, maps in the same region as rlx. The region of RK2 encompassing rlx has been cloned onto pBR322 and shown to promote mobilization of the hybrid plasmid by an RK2 derivative. These results indicate that the RK2 relaxation complex nicks at or near the origin of transfer of the RK2 plasmid.     

Detection and monitoring of anaerobic rumen fungi using an ARISA method

Aim: To develop an automated ribosomal intergenic spacer region analysis (ARISA) method for the detection of anaerobic rumen fungi and also to demonstrate utility of the technique to monitor colonization and persistence of fungi, and diet‐induced changes in community structure. Methods and Results: The method could discriminate between three genera of anaerobic rumen fungal isolates, representing Orpinomyces, Piromyces and Neocallimastix species. Changes in anaerobic fungal composition were observed between animals fed a high‐fibre diet compared with a grain‐based diet. ARISA analysis of rumen samples from animals on grain showed a decrease in fungal diversity with a dominance of Orpinomyces and Piromyces spp. Clustering analysis of ARISA profile patterns grouped animals based on diet. A single strain of Orpinomyces was dosed into a cow and was detectable within the rumen fungal population for several weeks afterwards. Conclusions: The ARISA technique was capable of discriminating between pure cultures at the genus level. Diet composition has a significant influence on the diversity of anaerobic fungi in the rumen and the method can be used to monitor introduced strains. Significance and Impact of the Study: Through the use of ARISA analysis, a better understanding of the effect of diets on rumen anaerobic fungi populations is provided.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

Genetic structure of natural populations of Nitrobacter in an aquatic environment

Nitrification has been essentially studied as a chemical process and the studies of the microbial population have been slightly worked out. This work constitutes a first attempt to study the diversity of Nitrobacter strains with the aim of working out the genetic structure of natural populations. The Nitrobacter population structure was compared between freshwater and sediments of the same lake. Nitrobacter isolates were identified and characterized by DNA/DNA hybridization, restriction pattern of rRNA genes, PCR/RFLP analysis of the ribosomal intergenic spacer and plasmid patterns. It was shown that this lake population was split into 3 subpopulations: a specific freshwater subpopulation, a specific sediment subpopulation and a nonspecific one. A 60 MDa plasmid was detected in 3,3% of freshwater isolates. A 37 MDa plasmid was detected in 59% of sediments isolates. This 37 MDa plasmid was not associated with a genomic group, but associated with a localization: the sediments. These results suggest that this 37 MDa plasmid could be implicated in the adaptation to sediment environment.     

Isolation of Bacillus megaterium Mutants That Produce High Levels of Heterologous Protein,and Their Use to Construct a Highly Mosquitocidal Strain

A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described [Appl Microbiol Biotechnol 35:594, 1991]. Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein. The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli. The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin. This strain shows toxicity to Culex quinquefasciatus larvae that is comparable to B. sphaericus 2362 and higher than a B. megaterium strain with the original expression plasmid. This approach may be generally useful for high-level regulated protein expression in B. megaterium. Received: 6 December 1996 / Accepted: 28 January 1997     

Use of a known plasmid and transposon as tracers for the incompatibility classification of a cryptic plasmid

Summary We have developed a novel genetic technique by which an unknown plasmid can be classified by the use of a plasmid of known incompatibility group. In phenocopy state, cells harboring plasmids of known incompatibility group behave as normal recipients and receive plasmids belonging to the same incompatibility group at a high frequency. This work provides additional evidence that plasmids in phenocopy hosts do not replicate and therefore fail to demonstrate their incompatibility barrier to the incoming plasmids. A cryptic plasmid, now called pWS7, has been identified by this method in a pili, fla female strain of E. coli K12. Genetic analysis shows the plasmid pWS7 is in fact, a sex-factor which is curable with acridine orange. It belongs to the Inc F1 group. Physical analysis confirms its size to be 124 Kb. The plasmid has been labelled genetically with a transposon Tn903 in a recA host and further characterized by heteroduplex analysis. A DNA sequence homology between pWS7 and F'lac plasmid extends only in F-regions, 2.8F-94.5F. The pili, fla host strain of pWS7 shows a high frequency of transformation for recombinant DNA and rapid propagation for a male-specific RNA phage, R17.