scholarly journals Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1

2012 ◽  
Vol 78 (23) ◽  
pp. 8254-8263 ◽  
Author(s):  
Michael Weiss ◽  
Karin Denger ◽  
Thomas Huhn ◽  
David Schleheck
Keyword(s):  
Degradation Pathway ◽  
Enzyme Hydrolysis ◽  
Transcriptional Analysis ◽  
Molar Ratio ◽  
Type I ◽  
Comamonas Testosteroni ◽  
Linear Alkylbenzenesulfonate ◽  
Content Type ◽  
Xenobiotic Compound ◽  
Enzyme Families

ABSTRACTComplete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized byComamonas testosteroniKF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.

scholarly journals Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire β-Oxidation Cycle of the Cleaved B Ring

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Masae Horinouchi ◽  
Hiroyuki Koshino ◽  
Michal Malon ◽  
Hiroshi Hirota ◽  
Toshiaki Hayashi
Keyword(s):  
Gene Clusters ◽  
Degradation Process ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Comamonas Testosteroni ◽  
Content Type ◽  
Coa Transferase ◽  
Degrading Bacteria ◽  
Globally Distributed

ABSTRACT Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7β,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters. IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire β-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

scholarly journals A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1

2017 ◽  
Vol 83 (23) ◽  
Author(s):  
Cui-Wei Chu ◽  
Bin Liu ◽  
Na Li ◽  
Shi-Gang Yao ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Microbial Degradation ◽  
Bond Cleavage ◽  
Degradation Pathway ◽  
Transcriptional Analysis ◽  
Flavin Mononucleotide ◽  
Monooxygenase System ◽  
Chlorobenzoic Acid ◽  
Content Type ◽  
Catabolic Genes ◽  
Two Component

ABSTRACT Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C—S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C—S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis. IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C—S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism.

scholarly journals The Missing Link in Linear Alkylbenzenesulfonate Surfactant Degradation: 4-Sulfoacetophenone as a Transient Intermediate in the Degradation of 3-(4-Sulfophenyl)Butyrate by Comamonas testosteroni KF-1

2009 ◽  
Vol 76 (1) ◽  
pp. 196-202 ◽  
Author(s):  
David Schleheck ◽  
Frederick von Netzer ◽  
Thomas Fleischmann ◽  
Daniel Rentsch ◽  
Thomas Huhn ◽  
...  
Keyword(s):  
Enzyme Reaction ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Comamonas Testosteroni ◽  
Linear Alkylbenzenesulfonate ◽  
Sulfite Oxidation ◽  
Surfactant Degradation ◽  
Transient Intermediates ◽  
Hydrolysis Of ◽  
Transient Intermediate

ABSTRACT Biodegradation of the laundry surfactant linear alkylbenzenesulfonate (LAS) involves complex bacterial communities. The known heterotrophic community has two tiers. First, all LAS congeners are oxygenated and oxidized to about 50 sulfophenylcarboxylates (SPC). Second, the SPCs are mineralized. Comamonas testosteroni KF-1 mineralizes 3-(4-sulfophenyl)butyrate (3-C4-SPC). During growth of strain KF-1 with 3-C4-SPC, two transient intermediates were detected in the culture medium. One intermediate was identified as 4-sulfoacetophenone (SAP) (4-acetylbenzenesulfonate) by nuclear magnetic resonance (NMR). The other was 4-sulfophenol (SP). This information allowed us to postulate a degradation pathway that comprises the removal of an acetyl moiety from (derivatized) 3-C4-SPC, followed by a Baeyer-Villiger monooxygenation of SAP and subsequent ester cleavage to yield SP. Inducible NADPH-dependent SAP-oxygenase was detected in crude extracts of strain KF-1. The enzyme reaction involved transient formation of 4-sulfophenol acetate (SPAc), which was completely hydrolyzed to SP and acetate. SP was subject to NADH-dependent oxygenation in crude extract, and 4-sulfocatechol (SC) was subject to oxygenolytic ring cleavage. The first complete degradative pathway for an SPC can now be depicted with 3-C4-SPC: transport, ligation to a coenzyme A (CoA) ester, and manipulation to allow abstraction of acetyl-CoA to yield SAP, Baeyer-Villiger monooxygenation to SPAc, hydrolysis of the ester to acetate and SP, monooxygenation of SP to SC, the ortho ring-cleavage pathway with desulfonation, and sulfite oxidation.

scholarly journals The Bacillus cereus Group Is an Excellent Reservoir of Novel Lanthipeptides

2014 ◽  
Vol 81 (5) ◽  
pp. 1765-1774 ◽  
Author(s):  
Bingyue Xin ◽  
Jinshui Zheng ◽  
Ziya Xu ◽  
Xiaoling Song ◽  
Lifang Ruan ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Gene Cluster ◽  
Transmembrane Protein ◽  
Gene Clusters ◽  
Transcriptional Analysis ◽  
Exponential Growth Phase ◽  
Type I ◽  
Structural Genes ◽  
Gram Positive ◽  
Content Type

ABSTRACTLantibiotics are ribosomally synthesized peptides that contain multiple posttranslational modifications. Research on lantibiotics has increased recently, mainly due to their broad-spectrum antimicrobial activity, especially against some clinical Gram-positive pathogens. Many reports about various bacteriocins in theBacillus cereusgroup have been published, but few were about lantibiotics. In this study, we identified 101 putative lanthipeptide gene clusters from 77 out of 223 strains of this group, and these gene clusters were further classified into 20 types according to their gene organization and the homologies of their functional genes. Among them, 18 types were novel and have not yet been experimentally verified. Two novel lantibiotics (thuricin 4A-4 and its derivative, thuricin 4A-4D) were identified in the type I-1 lanthipeptide gene cluster and showed activity against all tested Gram-positive bacteria. The mode of action of thuricin 4A-4 was studied, and we found that it acted as a bactericidal compound. The transcriptional analysis of four structural genes (thiA1,thiA2,thiA3, andthiA4) in the thuricin 4A gene cluster showed that only one structural gene,thiA4, showed efficient transcription in the exponential growth phase; the other three structural genes did not. In addition, the putative transmembrane protein ThiI was responsible for thuricin 4A-4 immunity. Genome analysis and functional verification illustrated thatB. cereusgroup strains were a prolific source of novel lantibiotics.

scholarly journals Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

2014 ◽  
Vol 197 (4) ◽  
pp. 749-761 ◽  
Author(s):  
M. A. Serbanescu ◽  
M. Cordova ◽  
K. Krastel ◽  
R. Flick ◽  
N. Beloglazova ◽  
...  
Keyword(s):  
Heat Shock ◽  
Streptococcus Mutans ◽  
Antibiotic Resistance Genes ◽  
Transcriptional Analysis ◽  
Cell Physiology ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Bacterial Physiology

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

scholarly journals Geographic Distribution of Secondary Metabolite Genes in the Marine Actinomycete Salinispora arenicola

2011 ◽  
Vol 77 (17) ◽  
pp. 5916-5925 ◽  
Author(s):  
Anna Edlund ◽  
Sandra Loesgen ◽  
William Fenical ◽  
Paul R. Jensen
Keyword(s):  
Secondary Metabolite ◽  
Geographic Distribution ◽  
Polyketide Synthase ◽  
Transcriptional Analysis ◽  
Type I ◽  
Molecular Fingerprinting ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Marine Actinomycete ◽  
Sea Of Cortez

ABSTRACTThe molecular fingerprinting technique terminal-restriction fragment length polymorphism (T-RFLP) was used in combination with sequence-based approaches to evaluate the geographic distribution of secondary metabolite biosynthetic genes in strains of the marine actinomyceteSalinispora arenicola. This study targeted ketosynthase (KS) domains from type I polyketide synthase (PKS) genes and revealed four distinct clusters, the largest of which was comprised of strains from all six global locations sampled. The remaining strains fell into three smaller clusters comprised of strains derived entirely from the Red Sea, the Sea of Cortez, or around the Island of Guam. These results reveal variation in the secondary metabolite gene collectives maintained by strains that are largely clonal at the 16S rRNA level. The location specificities of the three smaller clusters provide evidence that collections of secondary metabolite genes in subpopulations ofS. arenicolaare endemic to these locations. Cloned KS sequences support the maintenance of distinct sets of biosynthetic genes in the strains associated with each cluster and include four that had not previously been detected inS. arenicola. Two of these new sequences were observed only in strains derived from Guam or the Sea of Cortez. Transcriptional analysis of one of the new KS sequences in conjunction with the production of the polyketide arenicolide A supports a link between this sequence and the associated biosynthetic pathway. From the perspective of natural product discovery, these results suggest that screening populations from distant locations can enhance the discovery of new natural products and provides further support for the use of molecular fingerprinting techniques, such as T-RFLP, to rapidly identify strains that possess distinct sets of biosynthetic genes.

scholarly journals Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Leonardo Furi ◽  
Liam A. Crawford ◽  
Guillermo Rangel-Pineros ◽  
Ana S. Manso ◽  
Megan De Ste Croix ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lytic Replication ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Type I ◽  
Phase Variable ◽  
Abortive Infection ◽  
Content Type ◽  
Variable Type ◽  
Restriction Modification

ABSTRACTVirus-host interactions are regulated by complex coevolutionary dynamics. InStreptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of thenrdRnucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogenS. pneumoniae.IMPORTANCEWith antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogenStreptococcus pneumoniaeand explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

scholarly journals D-Ribose Catabolism in Archaea: Discovery of a Novel Oxidative Pathway in Haloarcula Species

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Ulrike Johnsen ◽  
Jan-Moritz Sutter ◽  
Andreas Reinhardt ◽  
Andreas Pickl ◽  
Rui Wang ◽  
...  
Keyword(s):  
Oxidative Degradation ◽  
Degradation Pathway ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Semialdehyde Dehydrogenase ◽  
Acid Dehydratase ◽  
Enolase Superfamily ◽  
Oxidative Pathway ◽  
Sugar Degradation

ABSTRACT The Haloarcula species H. marismortui and H. hispanica were found to grow on d-ribose, d-xylose, and l-arabinose. Here, we report the discovery of a novel promiscuous oxidative pathway of pentose degradation based on genome analysis, identification and characterization of enzymes, transcriptional analysis, and growth experiments with knockout mutants. Together, the data indicate that in Haloarcula spp., d-ribose, d-xylose, and l-arabinose were degraded to α-ketoglutarate involving the following enzymes: (i) a promiscuous pentose dehydrogenase that catalyzed the oxidation of d-ribose, d-xylose, and l-arabinose; (ii) a promiscuous pentonolactonase that was involved in the hydrolysis of ribonolactone, xylonolactone, and arabinolactone; (iii) a highly specific dehydratase, ribonate dehydratase, which catalyzed the dehydration of ribonate, and a second enzyme, a promiscuous xylonate/gluconate dehydratase, which was involved in the conversion of xylonate, arabinonate, and gluconate. Phylogenetic analysis indicated that the highly specific ribonate dehydratase constitutes a novel sugar acid dehydratase family within the enolase superfamily; and (iv) finally, 2-keto-3-deoxypentanonate dehydratase and α-ketoglutarate semialdehyde dehydrogenase catalyzed the conversion of 2-keto-3-deoxypentanonate to α-ketoglutarate via α-ketoglutarate semialdehyde. We conclude that the expanded substrate specificities of the pentose dehydrogenase and pentonolactonase toward d-ribose and ribonolactone, respectively, and the presence of a highly specific ribonate dehydratase are prerequisites of the oxidative degradation of d-ribose in Haloarcula spp. This is the first characterization of an oxidative degradation pathway of d-ribose to α-ketoglutarate in archaea. IMPORTANCE The utilization and degradation of d-ribose in archaea, the third domain of life, have not been analyzed so far. We show that Haloarcula species utilize d-ribose, which is degraded to α-ketoglutarate via a novel oxidative pathway. Evidence is presented that the oxidative degradation of d-ribose involves novel promiscuous enzymes, pentose dehydrogenase and pentonolactonase, and a novel sugar acid dehydratase highly specific for ribonate. This is the first report of an oxidative degradation pathway of d-ribose in archaea, which differs from the canonical nonoxidative pathway of d-ribose degradation reported for most bacteria. The data contribute to our understanding of the unusual sugar degradation pathways and enzymes in archaea.

scholarly journals Molecular Mechanisms ofhsdSInversions in thecodLocus ofStreptococcus pneumoniae

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Jing-Wen Li ◽  
Jing Li ◽  
Juanjuan Wang ◽  
Chunhao Li ◽  
Jing-Ren Zhang
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Phenotypic Diversity ◽  
Transcriptional Analysis ◽  
Inverted Repeats ◽  
Type I ◽  
Content Type ◽  
Dna Inversion ◽  
Methylation Patterns

ABSTRACTStreptococcus pneumoniae(pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferasehsdSgenes (hsdSA,hsdSB, andhsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all threehsdSgenes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression ofhsdSAbut nothsdSBandhsdSC, indicating thathsdSBandhsdSCdo not produce functional proteins and instead act as sources for altering the sequence ofhsdSAby DNA inversions. Consistent with our previous finding that thehsdSinversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion betweenhsdSAandhsdSB. Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in thepsrAmutant. Surprisingly, PsrA-independent inversions were also detected in thehsdSsequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed thatS. pneumoniaevaries the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in thecodlocus.IMPORTANCEStreptococcus pneumoniaeis a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferasehsdSgenes of thecodlocus. This work revealed that only thehsdSgene that is in the same orientation ashsdMis actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of thehsdSinversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed thatS. pneumoniaeswitches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

scholarly journals Modulation of Chicken Intestinal Immune Gene Expression by Small Cationic Peptides as Feed Additives during the First Week Posthatch

2013 ◽  
Vol 20 (9) ◽  
pp. 1440-1448 ◽  
Author(s):  
Michael H. Kogut ◽  
Kenneth J. Genovese ◽  
Haiqi He ◽  
Christina L. Swaggerty ◽  
Yiwei Jiang
Keyword(s):  
Gene Expression ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Feed Additives ◽  
Feed Additive ◽  
Cationic Peptides ◽  
Type I ◽  
Immune Gene ◽  
Content Type ◽  
Functional Efficiency

ABSTRACTWe have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium,Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activitiesin vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization bySalmonella entericaserovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and withoutS. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged withS. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection withS. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response toS. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection againstSalmonellainfections.

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