Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens.

AIMS: Determining the effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broilers. METHODS AND RESULTS: 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling, DNA sequencing and real-time quantitative PCR techniques were used. The richness of both ileal and caecal microbiota increased with chicken age. The microbiota from those birds of the same age demonstrated relatively similar PCR-DGGE profiles and tended to form closely related clusters in the relatedness analyses. Dietary treatment with bacitracin (50 mg kg(-1)) and access to range did not change the richness but altered the composition of the microbiota. The impact of bacitracin was particularly obvious in 3-day-old chicks. Lactobacilli were abundant in the caecal microbiota of 3-day-old chicks regardless of the dietary treatment with bacitracin. The access to range enriched Bifidobacterium in both the ileum and caeca. CONCLUSIONS: Bacitracin, bird age and access to range all influenced bacterial microbiota in the ileum and caeca of broilers, with bird age having the greatest apparent effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing useful information for the development of antibiotic replacement therapy for poultry production.

Characterization, nucleotide sequence, and conserved genomic locations of insertion sequence ISRm5 in Rhizobium meliloti.

A target for ISRm3 transposition in Rhizobium meliloti IZ450 is another insertion sequence element, named ISRm5. ISRm5 is 1,340 bp in length and possesses terminal inverted repeats of unequal lengths (27 and 28 bp) and contain five mismatches. An open reading frame that spans 89% of the length of one DNA strand encodes a putative transposase with significant similarity to the putative transposases of 11 insertion sequence elements from diverse bacterial species, including ISRm3 from R. meliloti. Multiple copies and variants of ISRm5 occur in the R. meliloti genome, often in close association with ISRm3. Five ISRm5 copies in two strains were studied, and each was found to be located between 8-bp direct repeats. At two of these loci, which were shown to be highly conserved in R. meliloti, the copies of ISRm5 were found to be associated with pairs of short inverted repeats resembling transcription terminators. This structural arrangement not only may provide a conserved niche for ISRm5 but also may be a preferred target for transposition.

Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium.

A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism. The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol. Strain ER2 contained two plasmids (120 and 130 kb). A probe cloned from the pDL11 plasmid of Achromobacter sp. strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P. H. Tomasek and J. S. Karns, J. Bacteriol. 171:4038-4044, 1989), hybridized to the 120-kb plasmid. Restriction fragment profiles of pDL11 and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11. On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogenetically unique type II methylotroph. The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose. Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation. These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment.

Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47.

The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.

Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm3: similarity between the putative transposase encoded by ISRm3 and those encoded by Staphylococcus aureus IS256 and Thiobacillus ferrooxidans IST2.

The insertion sequence ISRm3 was discovered simultaneously in different Rhizobium meliloti strains by probing Southern blots of total cellular DNA with 32P-labeled pTA2. This plasmid is indigenous to strain IZ450 and fortuitously contained four copies of ISRm3. By using an internal EcoRI fragment as a specific probe (pRWRm31), homology to ISRm3 was subsequently detected in over 90% of R. meliloti strains tested from different geographical locations around the world. The frequency of stable nonlethal ISRm3 transpositions was estimated to be 4 x 10(-5) per generation per cell in strain SU47 when grown in liquid culture. The entire nucleotide sequence of ISRm3 in R. meliloti 102F70 is 1,298 bp and has 30-bp terminal inverted repeats which are perfectly matched. Analysis of six copies of ISRm3 in two strains showed that a variable number of base pairs (usually eight or nine) were duplicated and formed direct repeats adjacent to the site of insertion. On one DNA strand, ISRm3 contains an open reading frame spanning 93% of its length. Comparison of the putative protein encoded with sequences derived from the EMBL and GenBank databases showed significant similarity between the putative transposases of ISRm3 from R. meliloti, IS256 from Staphylococcus aureus, and IST2 from Thiobacillus ferroxidans. These insertion sequences appear to be distantly related members of a distinct class.

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species.

Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1-3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A - H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B - H. leporinum; Map C - H. vulgare, H. spontaneum, H. agriocrithon; Map D - H. chilense, H. bogdanii; and Map E - remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

A Positive Strain Identification Method for Rhizobium meliloti.

About 80% of Rhizobium meliloti strains contain 1 to 11 copies of insertion sequence ISRm1 in their genomes (R. Wheatcroft and R. J. Watson, J. Gen. Microbiol. 134:113-121, 1988). Hybridization to separated genomic DNA fragments with an ISRm1-specific probe produces patterns of hybridization bands which are distinctive for each strain. These patterns can be compared between strains to prove or disprove common identity. In most cases relatedness can be inferred despite phenotypic differences or minor genomic alterations.

Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid.

A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria.

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.

Rapid methods for the study of both stable and unstable plasmids in Pseudomonas.

Rapid methods for the analysis of degradative plasmids in Pseudomonas are described. Bacterial lysates prepared with alkaline sodium dodecyl sulphate were vigorously stirred in the presence of antifoam agent. Samples were subjected to agarose gel electrophoresis to detect plasmid DNA. After a short period of centrifugation on a sucrose gradient, the lysates yielded plasmid DNA of sufficient purity for restriction endonuclease digestion without further treatment. The methods have proved most useful in the extraction of previously undetected plasmid DNA, some of which appears to be unstable.

Repair of UV-induced DNA damage and survival in yeast. I. Dimer excision.

The amount of pyrimidine dimer UV photoproduct lost from the DNA of irradiated yeast cells during dark incubation has been measured in various conditions. It was found that no dimers were lost when cells were incubated in saline. When the cells were incubated, with aeration, in a full growth medium, dimers were lost, most excision being complete within 4 h. Not all dimers were lost and the number lost was a function of UV dose. Maximum loss, amounting to 50 000 dimers per genome was observed after 4000 or 6000 erg/mm2 of UV. At higher doses, the number excised declined. Making the assumptions that dimers are the principal lethal product of UV, that a single dimer remaining in its genome is enough to prevent a cell from multiplying and that excision is the principal dark-repair process in yeast, these data were incorporated into the repair term of an expression relating survival to repair8 and it was found that the survival of yeast at doses up to 2000 erg/mm2 of UV could be quite accurately predicted. This is the first time it has been possible to account for survival in terms of measured repair. It is suggested that the divergence of the predicted and observed curves at higher doses is due to other processes known to exist in yeast.