High concentrations of tannins in fodder plants inhibit gastrointestinal bacteria and reduce ruminant performance. Increasing the proportion of tannin-resistant bacteria in the rumen protects ruminants from anti-nutritional effects. The reason for the protective effect is unclear, but could be elucidated if the mechanism(s) by which tannins inhibit bacteria and the mechanisms of tannin resistance were understood. A review of the literature indicates that the ability of tannins to complex with polymers and minerals is the basis of the inhibitory effect on gastrointestinal bacteria. Mechanisms by which bacteria can overcome inhibition include tannin modification/degradation, dissociation of tannin-substrate complexes, tannin inactivation by high-affinity binders, and membrane modification/repair and metal ion sequestration. Understanding the mechanism of action of tannins and the mechanism(s) bacteria use to overcome the inhibitory effects will allow better management of the rumen ecosystem to reduce the anti-nutritional effects of tannin-rich fodder plants and thereby improve ruminant production.
The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl--D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.
The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.Lyme disease, caused by Borrelia burgdorferi, is the most commonly reported arthropod-borne disease in both the United States and Europe (32). At the molecular level, certain membrane lipoproteins of B. burgdorferi are vital for maintaining the spirochete in its zoonotic transmission cycle between ticks and mammals. For example, the reciprocal regulation of outer surface (lipo)proteins A (OspA) and C (OspC) is the best-studied paradigm of the dramatic alterations in protein expression patterns that ensue as the spirochete transitions from the arthropod vector into mammalian tissues (8,22,30,31). Our lab determined previously that expression of OspC is regulated by the alternative sigma factor RpoS ( S ) (15, 40). RpoS, though, must first be activated by another alternative transcription factor, RpoN ( 54 / N ), resulting in an RpoNRpoS regulatory network (10, 15). However, the precise mechanism(s) by which RpoN activates rpoS has not been determined. RpoN could control rpoS expression directly by binding to a region near the rpoS open reading frame (ORF). Alternatively, another transactivator induced by RpoN might activate rpoS expression.We have been most attracted to the notion that RpoN binds directly to a region upstream of the rpoS ORF to facilitate RpoS expression in B. burgdorferi. This is because nucleotides Ϫ78 to Ϫ63 upstream of the rpoS ORF comprise a theoretical, RpoN-dependent consensus Ϫ24/Ϫ12 promoter (33, 34). Many studies with various bacteria have indicated close contact of RpoN with such a Ϫ24/Ϫ12 promoter region (5, 6, 21, 33). However, as yet, there have been no experimental data to substantiate this prediction for B. burgdorferi. The purpose of this study was to garner additional evidence that would either substantiate or refute the hypothesis that rpoS is regulated directly by RpoN.Identification of rpoS initiation of transcription. Determination of the initiation of transcription for a given gene can provide strategic information for identifying a gene's nearby promoter. rpoS transcripts of B. burgdorferi BbAH130 (41) were reverse transcribed in BD SMART-RACE (switching mechanism at 5Ј end of RNA transcript-rapid amplification of cDNA ends) reactions (BD Biosciences, San Jose, CA) using two rpoS-specific primers (rpoSR422 and rpoSR232) (Table 1), which are 422 and 232 bases, respectively, downstream of the rpoS translation start site. The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3Ј end of the first-strand cDNA, and an rpoS-specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) ( Table 1) were used to amplify the resulting cDNAs. PCR-amplified inserts clo...
The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tanninresistant bacteria increased significantly (P < 0.05) from 0.3% ؎ 5.5% to 25.3% ؎ 8.3% with a 0.7% tannin diet and to 47.2% ؎ 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species.
The objective of this study was to investigate the effects of dietary Bacillus subtilis supplementation on growth performance, jejunal lesion scores, oocyst shedding, and cytokine and tight junction protein expression in broiler chickens infected with Eimeria maxima . A total of 196 male day-old Ross 708 broilers were given a nonexperimental diet until 14 D of age. Then, all chickens were randomly assigned to one of seven dietary treatments: 2 basal diets ( CON and NC ); CON + virginiamycin ( AB1 ); CON + bacitracin methylene disalicylate ( BMD ; AB2 ); CON + B. subtilis 1781 ( PB1 ); CON + B. subtilis 747 ( PB2 ); or CON + B. subtilis 1781 + 747 ( PB3 ). At day 21, all chickens except those in the CON group were orally inoculated with E. maxima oocysts. At 7 D after E. maxima infection, the body weight gains of chickens fed PB2 and PB3 increased ( P = 0.032) as much as those in chickens fed AB2. The body weight gain and feed efficiency of chickens fed PB2 were significantly increased ( P < 0.001), and PB2 chickens showed ( P = 0.005) the lowest lesion scores after E. maxima infection. Chickens fed PB2 showed ( P < 0.05) lower mRNA expression of IL-1β in infected chicken groups. Chickens in the AB1, AB2, PB1, PB2, and PB3 groups showed ( P < 0.05) greater mRNA expression of junctional adhesion molecule 2 in jejunal tissue, whereas occludin expression increased ( P < 0.05) in the jejunal tissue of chickens fed AB2 or PB2. Dietary B. subtilis supplementation significantly improved the growth performance of young chickens to a level comparable with that induced by virginiamycin or BMD without E. maxima infection. After infection with E. maxima , dietary virginiamycin and BMD significantly enhanced the epithelial barrier integrity, and the dietary B. subtilis 747 showed significantly enhanced growth performance, intestinal immunity, and epithelial barrier integrity. Together our results indicated that certain strains of B. subtilis provide beneficial effects on the growth of young broiler chickens and have the potential to replace antibiotic growth promoters.
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