Identification of proteolytic rumen bacteria isolated from New Zealand cattle

The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured. Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis. Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens , while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains. Further tests identified strains of cluster B as Eubacterium budayi , while cluster D strains most closely resembled B. fibrisolvens and were described as B. fibrisolvens -like. An unclustered strain, C21a, was identified as P. ruminicola. The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.     

Intracellular nuclease activities of buffalo rumen bacterial population

Nuclease activities of the predominantly bacterial population obtained from buffalo rumen were investigated. Optimum temperature for hydrolysis of both DNA and RNA was 50°C whereas DNAase activity was observed to be stable up to 50°C, a decrease in RNAase activity was observed even after 40°C. Two pH optima, one at 5.5 and the other at 7.5, were recorded for hydrolysis of DNA. RNAase activity was maximum between pH 6.0 to 7.0. Whereas DNAase activity was stable near its optimum pH, RNAase activity was stable between pH 7.0 to 8.5. Mn²⁺ ions stimulated DNAase activity. It was strongly inhibited by Hg²⁺, Zn²⁺, Pb²⁺ and Ag⁺. RNAase activity was stimulated by Mg²⁺ ions and was strongly inhibited by Hg²⁺, Cu²⁺, Zn²⁺ and Ag⁺. Cysteine hydrochloride and 2-mercaptoethanol stimulated DNAase activity. The activity was strongly inhibited by N-ethylmaleimide, 4-chloromercuribenzoate, 8-quinolinol, iodoacetic acid and 1,10-phenanthroline. RNAase activity was stimulated by cysteine hydrochloride, reduced glutathione and 2-mercaptoethanol and was strongly inhibited by 4-chloromercuribenzoate, 8-quinolinol and 2,2′-bipyridyl. Part of PhD Thesis submitted by the first author to Kurukshetra University.     

Characterization of rumen bacterial strains isolated from enrichments of rumen content in the presence of propolis

Propolis presents many biological properties, including antibacterial activities, and has been proposed as an additive in ruminant nutrition. Twenty bacterial strains, previously isolated from enrichments of Brazilian cow rumen contents in the presence of different propolis extracts (LLOS), were characterized using phenotyping and 16S rRNA identification. Seven strains were assigned to Streptococcus sp., most likely S. bovis, and were all degrading starch. One amylolytic lactate-utilizing strain of Selenomonas ruminantium was also found. Two strains of Clostridium bifermentans were identified and showed proteolytic activity. Two strains were assigned to Mitsuokella jalaludinii and were saccharolytic. One strain belonged to a Bacillus species and seven strains were affiliated with Escherichia coli. All of the 20 strains were able to use many sugars, but none of them were able to degrade the polysaccharides carboxymethylcellulose and xylans. The effect of three propolis extracts (LLOS B1, C1 and C3) was tested on the in vitro growth of four representative isolates of S. bovis, E. coli, M. jalaludinii and C. bifermentans. The growth of S. bovis, E. coli and M. jalaludinii was not affected by the three propolis extracts at 1 mg ml^?1. C. bifermentans growth was completely inhibited at this LLOS concentration, but this bacterium was partially resistant at lower concentrations. LLOS C3, with the lower concentration of phenolic compounds, was a little less inhibitory than B1 and C1 on this strain.     

Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage

Aims: To determine the effects of the removal of forage in high‐concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture‐independent methods. Methods and Results: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson’s index of 16S PCR‐DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real‐time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet. Conclusions: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined. Significance and Impact of the Study: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.     

Antagonistic and antimicrobial activities of some bacterial isolates collected from soil samples

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunflower, wilts and root rots, respectively. Among them, 5 bacterial strains, viz., A1 6 (Bacillus sphaericus), K1 24 (Pseudomonas fluorescens), M1 42 (Bacillus circulans), M1 66 (Bacillus brevis) and T1 22 (Bacillus brevis) showed positive antagonistic activity. M1 66 was the most effective in inhibiting mycelial growth of S. rolfsii in vitro followed by M1 42, T1 22, K1 24 and A1 6. Only one bacterial strain i.e. M1 42 exhibited antagonistic activity against F. oxysporum, and none of the bacterial strains gave positive activity against R. solani. Furthermore, antimicrobial activities of all the 5 strains were checked against different test organisms. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain.     

Characterization of proteolytic activities in embryos of Xenopus laevis

1. Proteolytic activities in early embryos of Xenopus laevis exhibited maximum levels at pH 3.2, 5.6 and 7.2 when 3H-BSA was used as substrate, and the maximum proteolytic activity at pH 3.2 was several thousand-fold higher during the tail bud stage than in the unfertilized egg. 2. The proteolytic activity at pH 3.2 was separated into two fractions by gel chromatography. One fraction corresponded to a mol. wt of about 40,000 and its activity was inhibited by thiol protease inhibitors. The other appeared to be a protease of much higher mol. wt. 3. The maximum activities at pH 5.6 and 7.2 appear to correspond to proteins of mol. wt greater than 1,000,000.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Characterization and crop production efficiency of diazotrophic bacterial isolates from coastal saline soils

Use of eco-friendly area specific salt tolerant bioinoculants is better alternatives to chemical fertilizer for sustainable agriculture in coastal saline soils. We isolated diverse groups of diazotrophic bacteria from coastal saline soils of different forest and agricultural lands in the Sundarbans, West Bengal, India, to study their effect on crop productivity in saline soils. Phenotypic, biochemical and molecular identifications of the isolates were performed. The isolates produced indole acetic acid, phosphatase, and solubilized insoluble phosphates. Sequence analysis of 16S rDNA identified the SUND_BDU1 strain as Agrobacterium and the strains SUND_LM2, Can4 and Can6 belonging to the genus Bacillus. The ARA activity, dinitrogen fixation and presence of nifH genes indicated they were diazotrophs. Field trials with these strains as bioinoculants were carried out during 2007-2009, with rice during August-December followed by Lady's finger during April-June. Microplots, amended with FYM inoculated with four bioinoculants individually were compared against sole FYM (5 t ha(-1)) and a sole chemical fertilizer (60:30:30 kg ha(-1) NPK) treated plot. The strain Can6 was by far the best performer in respect of yield attributes and productivity of studied crops.     

Characterization of a small proteolytic enzyme which lyses bacterial cell walls

Ensign, J. C. (University of Wisconsin, Madison), and R. S. Wolfe. Characterization of a small proteolytic enzyme which lyses bacterial cell walls. J. Bacteriol. 91:524-534. 1966.-An enzyme isolated from a myxobacter possesses both cell-wall lytic and proteolytic activity. The enzyme has been purified over 600-fold and is electrophoretically homogeneous upon cellulose acetate at several pH values and upon polyacrylamide gel columns. A single peak was obtained upon ultracentrifugation and density gradient centrifugation. Based upon Sephadex gel filtration, a molecular weight of 8,700 was determined for the enzyme. Albumin and casein were extensively degraded by the enzyme, with approximately one-third of the peptide bonds present in these proteins being hydrolyzed. The enzyme lyses cell walls by hydrolyzing peptide bonds in the glycosaminopeptide.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4°C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography [1]. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoproteins by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by and alkaline pH optimum and an estimated molecular mass of 80–100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 μM. The retained material eluted by addition of 1% methyl-α-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (> 70 kDa) and the other peak of lower molecular mass (30–70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Intimin subtyping of Escherichia coli: concomitant carriage of multiple intimin subtypes from forage-fed cattle and sheep

The outer membrane protein, intimin ( eae ), which mediates bacterial attachment to epithelial cells, is associated with enteropathogenic Escherichia coli and some Shiga toxin-producing E. coli . The eae subtype of E. coli strains isolated from healthy cattle and sheep was identified using a rapid PCR-restriction fragment length polymorphism (RFLP) method to produce profiles that were compared with those generated in silico . The 139 eae -positive E. coli strains were separated into 11 different PCR-RFLP profiles. The most common eae PCR-RFLP type was β (23.7%), followed by ζ (20.1%), θ (16.5%), ι (12.2%), κ (8.6%), ɛ (7.2%), γ (2.9%), ν and β2 (2.2%) and ι2 (1.4%). Four isolates did not yield a PCR-RFLP amplification product but complete sequencing of the eae gene matched subtype ρ. Two different eae variants were isolated from the same swab from 18 different animals and subtype ι was the most 'promiscuous', being isolated with four other eae subtypes from seven separate animals. None of the eae -positive STEC were subtype γ, which is associated with STEC serogroup O157. This method allowed the rapid identification of eae subtypes and indicates that forage-fed animals possessed a wide diversity of bacterial eae subtypes with a low frequency of eae subtype γ.     

Culture conditions influencing phytase production of Mitsuokella jalaludinii,a new bacterial species from the rumen of cattle

AIMS: To determine the effectiveness of combined treatments with chemicals, heat and ultrasound in killing or removing Salmonella and Escherichia coli O157:H7 on alfalfa seeds intended for sprout production. METHODS AND RESULTS: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 were treated with ultrasound (38.5-40.5 kHz) in solutions containing 1% Ca(OH)(2), 1% Tween 80, 1% Ca(OH)(2) plus 1% Tween 80, 160 microg ml(-1) Tsunami 200 and 0.5% Fit at 23 and 55 degrees C for 2 and 5 min. Highest reductions were in chemical solutions at 55 degrees C, but seed viability was also reduced compared with treatment at 23 degrees C. Inactivation of Salmonella and E. coli O157:H7 was generally enhanced by simultaneous treatments with ultrasound, chemicals and heat. CONCLUSIONS: Ultrasound treatment, in combination with chemicals and heat, had a modest enhancing effect on the effectiveness of chemicals in killing or removing pathogens on alfalfa seeds. Overall, treatment with 1% Ca(OH)(2) was most effective in killing Salmonella and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of 1% Ca(OH)(2) instead of 20,000 microg ml(-1) chlorine, which is currently recommended as a sanitizer for seeds intended for sprout production in the US, should be considered. Ultrasound treatment of alfalfa seeds containing Salmonella or E. coli O157:H7, in combination with chemical treatment, contributes to achieving greater reductions in populations of these pathogens, thereby reducing the risk of contamination and the presence of pathogens in sprouts produced from these seeds.     

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.     

Synergism between different species of proteolytic rumen bacteria

Proteolytic strains ofButyrivibrio alactacidigens, Butyrivibrio fibrisolvens, Selenomonas ruminantium, andStreptococcus bovis grew markedly better in medium containing casein as sole nitrogen source when they were inoculated in pairs rather than singly, when growth was poor or nil. The improved growth ofSelenomonas ruminantium with other species was a consequence of a faster rate of proteolysis by the mixed culture. The cooperativity withStreptococcus bovis was mediated by the extracellular activity ofSelenomonas ruminantium. Synergistic growth betweenBu. alactacidigens and other bacteria was not due to cooperativity between proteolytic activities but probably to nutritional interdependence between the species. Cooperative proteolysis also occurred in mixtures ofSelenomonas ruminantium andBacteroides ruminicola, a proteolytic isolate capable of growth on casein in monoculture.     

In vitro activities of antimicrobials against Brucella abortus isolates from cattle in Korea during 1998-2006

In vitro activities of 13 antibiotics were assessed against 85 Brucella abortus isolates from naturally infected cattle in the Republic of Korea during 1998-2006, using broth microdilution test. Tetracyclines showed the most excellent activity against B. abortus, displaying MIC values of 0.5 μg/ml or below. In particular, minocycline showed the lowest MIC??/?? values (0.125/0.125 μg/ml) in this study. Among four fluoroquinolones tested, ciprofloxacin (MIC??/??, 0.5/1 μg/ml) and norfloxacin (MIC??/??, 8/8 μg/ml) had the most and the least activities, respectively. Gentamicin (MIC??/??, 1/1 μg/ml) was more effective than streptomycin, erythromycin, rifampin, and chloramphenicol (MIC??/??, 2/2 μg/ml).     

Characteristics of isolates of Lactobacillus fermentum from the rumen of sheep

A detailed characterization of four representative Lactobacillus isolates from the rumen was undertaken after tests with API CH50 kits had indicated that they differed from currently recognized species. These isolates are shown to be related to Lactobacillus fermentum , although they differ from the type and reference strains of this species in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns and electrophoretic mobility of LDH. It seemed that certain heterofermentative lactobacilli could give negative results for some fermentation tests (particularly fructose and lactose) in API kits, whilst showing good growth on the same substrates in conventional tests.