Heat and desiccation are the predominant factors affecting inactivation of Bacillus licheniformis and Bacillus thuringiensis spores during simulated composting.

AIMS: The suitability of composting for disposal of livestock mortalities due to Bacillus anthracis was assessed by measuring viability of surrogate spores from two strains each of Bacillus licheniformis and Bacillus thuringiensis after a heating cycle modelled on a cattle composting study. METHODS AND RESULTS: Sporulation was attempted from 10 to 37°C, but poor yields at lower temperatures resulted in 25, 30 and 37°C being selected to generate sufficient spores (8 log10  CFU ml(-1) ) for experiments. Spores were inoculated into 3 g autoclaved dried-ground compost rehydrated with 6 ml water or silica beads in a factorial design for each strain, sporulation temperature, matrix and sampling day (0, 25, 50, 100, 150). Maximum incubation temperature was 62°C, but spores were maintained at ≥55°C for 78 of 150 days. Although significant differences existed among Bacillus strains and sporulation temperatures, numbers of viable spores after 150 days averaged 1·3 log10  CFU g(-1) , a 5·2 log10 reduction from day 0. CONCLUSIONS: Spore inactivation was likely due to heat and desiccation as matrices were autoclaved prior to incubation, negating impacts of microflora. SIGNIFICANCE AND IMPACT OF STUDY: Results support composting for disposal of anthrax mortalities, provided long-term thermophillic heating is achieved. Due to limited sporulation at 10°C, livestock mortalities from anthrax at this or lower ambient temperatures would likely be of lower risk for disease transmission.

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity.

AIMS: This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle. METHODS AND RESULTS: Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae- or Siphoviridae-like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·0001). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70-79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2-like phage φMhaA1-PHL101. CONCLUSIONS: Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.

Effects of temperature on mortality of larval stable fly (Diptera: Muscidae) caused by five isolates of Bacillus thuringiensis.

We examined the effects of temperature on mortality of larval stable fly [Stomoxys calcitrans (L.)] caused by Bacillus thuringiensis tolworthi 4L3, B. t. darmastedensis 4M1, B. t. thompsoni 401, B. t. thuringiensis HD2, and B. t. kurstaki HD945. At moderate doses, mortality caused by all isolates ranged from 87 to 99% at 15 degrees C and declined to 29-63% as temperature increased to 30 degrees C. A similar pattern was seen when a higher dose was used, except that the reduction in mortality at warmer temperatures was not as great as was seen with the moderate doses. Insecticidal activity of each isolate against first-instar larvae was reduced by only 5-15% after 5 d in the medium. Mortality of second- and third-instar larvae ranged from 2 to 21%, suggesting the isolates were less effective against larger larvae.

Mortality of adult Stomoxys calcitrans fed isolates of Bacillus thuringiensis.

We examined the ability of five isolates of Bacillus thuringiensis Berliner to cause mortality in adult stable flies, Stomoxys calcitrans (L.). Isolates Bacillus thuringiensis tolworthi 4L3 (serotype 9), Bacillus thuringiensis darmstadiensis 4M1 (serotype 10a10b), Bacillus thuringiensis thompsoni 401 (serotype 12), Bacillus thuringiensis thuringiensis HD2 (serotype 1), and Bacillus thuringiensis kurstaki HD945 (serotype 3a3b3c) were administered to adult flies in diets containing blood only, sugar only, and both sugar and blood combined. B. t. tolworthi 4L3 had no effect on adult mortality regardless of the feeding substrate. The remaining isolates tended to cause the greatest mortality when administered in blood alone. B. t. thompsoni 401 was the only isolate that consistently caused adult mortality when fed in blood at concentrations ranging from 0.21 to 50.0 microg of protein per ml of blood. This isolate also caused mortality when applied topically. The time to 50% mortality declined with dose and reached a lower asymptote at approximately equal to 1.3 d at an oral dose of 8.75 microg/ml and at a topical dose of 0.14 microg per fly.

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus.

AIMS: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. CONCLUSIONS: Fae1A exhibited a lower K(m) and higher catalytic efficiency (k(cat) /K(m) ) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes.

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).

Activity of Bacillus thuringiensis isolates against immature horn fly and stable fly (Diptera: Muscidae).

We screened 85 isolates of Bacillus thuringiensis (Berliner), making up 57 different subspecies, and two isolates of Bacillus sphaericus (Meyer and Neide) for activity against immature horn flies, Haematobia irritans (L.), and stable flies, Stomoxys calcitrans (L.). The majority of B. thuringiensis and the B. sphaericus isolates had little or no activity against horn fly and stable fly. Approximately 87% of the isolates caused < 50% mortality of horn fly larvae and 64% caused < 25% mortality. For stable fly, 95% of the isolates caused < 50% mortality, and 93% caused < 25% mortality. Five isolates were highly toxic to horn fly and stable fly immatures. These isolates were B. t. tolworthi 4L3, B. t. darmstadiensis 4M1, B. t. thompsoni 401, B. t. thuringiensis HD2, and B. t. kurstaki HD945. The LD50 values ranged from 2.2 to 7.9 x 10(6) spores per g manure for horn fly and from 6.3 to 35 x 10(6) spores per g media for stable fly. These were consistently more toxic compared with the B. t. israelensis isolates examined. All had DNA that hybridized with cry1Aa, cry1Ab, and cry1Ac toxin probes, three hybridized with a cry1B probe, and two hybridized with a cry2A probe. These may have potential for use in integrated management of pest flies.

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida.

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24-70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250-430) likely forms a 16-strand beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.

Retention of Escherichia coli by house fly and stable fly (Diptera: Muscidae) during pupal metamorphosis and eclosion.

Populations of Escherichia coli obtained by feeding larval house flies, Musca domestica L. and stable flies, Stomoxys calcitrans (L.), persisted through the pupal stage. The abundance of E. coli in house fly pupae increased initially then declined before adult emergence. Abundance of E. coli in stable fly pupae increased through pupal development and remained high. Infected stable fly pupal cases typically contained more E. coli than house fly pupal cases. A greater proportion of emerging adult house flies were infected with E. coli compared with stable flies; however, the abundance of E. coli on infected flies was similar between species. Adult flies contained 0.04-0.19% of the E. coli in the pupal cases. The proportion of infected house fly adults and the amount of E. coli on the infected flies were related to the levels of E. coli in the pupal cases; however, these relationships did not occur with the stable fly. Results suggest that retention of E. coli from larval to adult house flies could play a role in the transmission and spread of E. coli, whereas stable fly adults probably play a minor role in E. coli spread. However, pupae of both species have potential to act as reservoirs for E. coli.

Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.

A novel staining method for detecting phytase activity.

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

Persistence of Escherichia coli in immature house fly and stable fly (Diptera: Muscidae) in relation to larval growth and survival.

The persistence of Escherichia coli in artificially fed larvae was examined for up to 48 h after ingestion by house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). The rate of change in the E. coli load was similar for both species for up to 5 h after ingestion. Up to 48 h after ingestion, abundance of E. coli declined in immature house flies but remained constant in immature stable flies. When different E. coli concentrations were fed to larvae, the abundance of E. coli increased in stable fly larvae regardless of the initial concentration. The E. coli load in house fly larvae increased when larvae were fed a low concentration of bacteria, but it declined when larvae were fed a high concentration of bacteria. Survival of house fly and stable fly larvae averaged 62 and 25%, respectively, when reared on pure E. coli cultures. These observations suggest that house fly larvae digest E. coli and use it as a food source but stable fly larvae do not.

Comparsion of selected growth media for culturing Serratia marcescens, Aeromonas sp., and Pseudomonas aeruginosa as pathogens of adult Stomoxys calcitrans (Diptera: Muscidae).

Stable flies, Stomoxys calcitrans (L.), were orally infected with Aeromonas sp., Pseudomonas aeruginosa (Schroeter), and Serratia marcescens Bizio that were cultured on egg-yolk media, nutrient broth, and fly egg media. Aeromonas and Serratia caused mortality when the bacteria were originally grown on egg-yolk medium. Pseudomonas was equally lethal regardless of the media on which it was cultured. A wild isolate of Aeromonas caused greater death than an isolate that had been passed through host flies and had been reisolated from killed flies. Mortality increased with bacterial dose for all species. Aeromonas and Serratia caused mortality within several days after ingestion, whereas Pseudomonas caused a gradual increase in mortality 3-7 d after ingestion. The pathologic activity of Aeromonas and Serratia required extracellular products produced when cells were grown in egg yolk medium. Aeromonas required both supernatant and cells from egg yolk medium, wereas Serratia required supernatant from egg yolk medium and cells from either nutrient broth or egg yolk medium. Mortality due to ingestion of Aeromonas was correlated with the presence of enzymes that cause alpha- and beta-hemolysis, while mortality following ingestion of Serratia was associated with alpha-hemolysins, elastases, and chitinases.

Growth and survival of immature Heamatobia irritans (Diptera; Muscidae) is influenced by bacterial isolated from cattle manure and conspecific larvae.

Twenty species of bacteria were isolated from cattle manure and seven species were isolated from the gut of larval horn fly Hematobia irritans (L.). Bacteria in manure belonged to the Bacillaceae, Pseudomonadaceae, Micrococcaceae, Corynebacteriaceae, Enterobacteriaceae, Microbacteriaceae, and two unassigned genera. Gut bacteria belonged to the Enterobacteriaceae, Bacillaceae, Neisseriaceae, and Pseudomonadaceae. H. irritans larval survival and growth on the various bacterial species were evaluated by rearing larvae in sterilized cattle manure that was inoculated with single bacterial isolates. H. irritans larvae failed to develop in sterilized, uninoculated manure, indicating that bacteria are necessary for larval development. Survival averaged 74% in nonsterilized manure and ranged from 4 to 53% in manure with individual isolates. Survival was highest when larvae were reared on manure inoculated with Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, and Bacillaceae and was lowest when reared in manure inoculated with Enterobacteriaceae and Microbacteriaceae. Pupal weights were heaviest when reared on the Flavobacteria, followed by the Pseudomonadaceae and Corynebacteriaceae. Pupae averaged 4.9 +/- 0.08 mg when reared on gram-negative isolates, compared with 3.6 +/- 0.09 mg when reared on gram-positive isolates. Pupal weights were not significantly correlated with larval survival, indicating that bacteria that promote growth do not necessarily promote survival. A reproductive index was used as a measure of fitness and was highest for larvae reared in the nonsterile control, followed most closely by Pseudomonadaceae and Corynebacteriaceae. These groups appeared to best meet the nutritional requirements of larvae and may be used in further experiments to define an artificial rearing media for H. irritans.

Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.

The growth and survival of Stomoxys calcitrans (L.) larvae on egg yolk medium inoculated with bacteria isolated from a colony of stable flies was evaluated. Five species of bacteria--Acinetobacter sp., Aeromonas sp., Empedobacter breve (Holmes & Owen), Flavobacterium odoratum Stutzer, and Serratia marcescens Bizio--were identified according to fatty acid profiles using a microbial identification system. Larvae failed to develop on uninoculated plates, confirming that bacteria are required to complete development. Larvae also failed to complete development on plates inoculated with Aeromonas sp. and S. marcescens, and died during the 1st instar. Larvae completed development on the remaining 3 bacterial species as well as on Escherichia coli (Migula). Survival was generally higher when larvae were reared on Acinetobacter sp. and F. odoratum compared with E. coli and E. breve. Egg density did not influence larval survival, although the variability in survival was lowest using 20 and 40 eggs per plate. Larval survival in mixed cultures of Acinetobacter and Flavobacterium averaged 22.7% lower than survival in the pure cultures, and averaged 21.6% higher in mixed cultures of Empedobacter and Flavobacterium compared with pure cultures. Larval survival in mixed cultures did not differ significantly from mean survival in pure cultures for combinations of Acinetobacter and E. coli, Acinetobacter and Empedobacter, E. coli and Empedobacter, and E. coli and Flavobacterium. Larval developmental time was faster on all mixed bacterial cultures compared with developmental time on pure bacterial cultures. Optimal sample sizes and egg numbers are presented for detecting specified differences in larval survival. This rearing procedure will be useful for studying insect-microbe interactions and evaluating mortality using bacterial agents.

Genetic characterization and antimicrobial susceptibility of Mannheimia haemolytica isolated from the nasopharynx of feedlot cattle.

A surveillance study was undertaken to examine the population dynamics and antimicrobial resistance of Mannheimia haemolytica isolated from feedlot cattle. A total of 416 isolates were collected from the nasopharynx either upon entry or exit from two feedlots in southern Alberta, Canada. Isolates were serotyped, characterized by pulsed-field gel electrophoresis and tested for susceptibility to ten antimicrobial agents via disk diffusion. Resistant isolates were screened by PCR for select antimicrobial-resistance gene determinants. Isolates were highly diverse, with 335 unique pulsed-field profiles identified among 147 strongly related clusters (similarity ≥ 85%). Clonal spread of isolates throughout the feedlots was limited and no clear association was found between genetic relatedness of M. haemolytica and sampling event (entry or exit). Pulsed-field profiles sharing a common serotype and resistance phenotype tended to cluster together. The majority of isolates were identified as serotype 2 (74.5%) although both serotype 1 (11.9%) and 6 (12.7%) were detected. Only 9.54% of isolates exhibited antimicrobial resistance. Resistance to oxytetracycline was most prevalent (n=16), followed by ampicillin (n=10), and amoxicillin/clavulanic acid (n=7). Multi-drug resistance was observed in five isolates. The tetH gene was detected in all but two oxytetracycline resistant isolates. Other detectable resistance determinates included ermX and bla(ROB-1). In the two feedlots examined, M. haemolytica exhibited considerable genetic diversity and limited resistance to common veterinary antibiotics. Garnering further information on the linkage between genotype and phenotype should contribute toward a better understanding of the pathogenesis and dissemination of M. haemolytica in feedlots.

Comparison of repetitive PCR and pulsed-field gel electrophoresis for the genotyping of Mannheimia haemolytica.

Mannheimia haemolytica is an opportunistic pathogen that can cause fibrinonecrotic pneumonia in cattle and is the main bacterial agent implicated in bovine respiratory disease-complex (BRD). Despite its economic importance to the cattle industry, few studies have characterized the genetic nature of M. haemolytica and none have genotyped isolates from feedlots. Identifying and monitoring genetic variants of M. haemolytica is important to understanding the etiology of BRD in cattle. We investigated the capacity of three genotyping techniques (BOX-PCR, (GTG)(5)-PCR and PFGE analysis of SalI-restricted DNA) to discriminate among 24 reference strains from the family Pasteurellaceae and 40 M. haemolytica isolates collected from feedlot cattle. From cluster analysis of the M. haemolytica isolates, PFGE was revealed as most discriminating, followed by BOX-PCR and then (GTG)(5)-PCR (Simpson's diversity index >0.98, 0.82, and 0.72, respectively). Of these methods, PFGE also had the greatest mean repeatability (0.96). The PFGE and BOX-PCR assays grouped all M. haemolytica in a single cluster but only BOX-PCR and (GTG)(5)-PCR grouped the Mannheimia glucosida and Mannheimia ruminalis strains together. Refinement of genotyping procedures for M. haemolytica could offer new insight into the etiology of this pathogen in BRD.

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM.

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.

The rumen: a unique source of enzymes for enhancing livestock production.

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of transgenic plants expressing fibrolytic or phytase activity and of transgenic mice able to produce endoglucanase in the pancreas speak to the feasibility of improving feed digestion through genetic modification of the feedstuffs and the animals.

A conjugative transfer system for the rumen bacterium, Butyrivibrio fibrisolvens, based on Tn916-mediated transfer of the Staphylococcus aureus plasmid pUB110.

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 x 10(-7) per donor and 1.11 x 10(-5) per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 x 10(-6) per donor and 4.70 x 10(-8) per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.