scholarly journals Identification and Molecular Characterization of the Chromosomal Exopolysaccharide Biosynthesis Gene Cluster from Lactococcus lactis subsp. cremoris SMQ-461

2005 ◽  
Vol 71 (11) ◽  
pp. 7414-7425 ◽  
Author(s):  
N. Dabour ◽  
G. LaPointe
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Repeat Unit ◽  
Raw Milk ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Biosynthesis Gene Cluster ◽  
Glycosyltransferase Gene ◽  
Reverse Transcription Pcr ◽  
Exopolysaccharide Biosynthesis

ABSTRACT The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 × 106 Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5′ end and orfY, along with an IS981-related tnp gene, at the 3′ end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.

scholarly journals Characterization of the Role of para-Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

2007 ◽  
Vol 73 (8) ◽  
pp. 2673-2681 ◽  
Author(s):  
Arno Wegkamp ◽  
Wietske van Oorschot ◽  
Willem M. de Vos ◽  
Eddy J. Smid
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Gene Clusters ◽  
Defined Medium ◽  
Biosynthesis Pathway ◽  
Chemically Defined Medium ◽  
Aminobenzoic Acid ◽  
Biosynthesis Gene Cluster ◽  
Gene Coding ◽  
Aba Biosynthesis

ABSTRACT The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated, but detailed analysis revealed that pabC was fused with the 3′ end of the gene coding for chorismate synthetase component II (pabB). Therefore, we hypothesize that all three enzyme activities needed for pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabB C was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and pABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without pABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals Molecular characterization of the plasmid‐encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis

1997 ◽  
Vol 24 (2) ◽  
pp. 387-397 ◽  
Author(s):  
Richard van Kranenburg ◽  
Joey D. Marugg ◽  
Iris I. Van Swam ◽  
Norwin J. Willem ◽  
Willem M. De Vos
Keyword(s):  
Gene Cluster ◽  
Molecular Characterization ◽  
Lactococcus Lactis ◽  
Exopolysaccharide Biosynthesis

scholarly journals A Putative C2H2 Transcription Factor CgTF6, Controlled by CgTF1, Negatively Regulates Chaetoglobosin A Biosynthesis in Chaetomium globosum

2021 ◽  
Vol 2 ◽  
Author(s):  
Yu Yan ◽  
Biyun Xiang ◽  
Qiaohong Xie ◽  
Yamin Lin ◽  
Guangya Shen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Gene Cluster ◽  
Transcriptional Activation ◽  
Chaetomium Globosum ◽  
Regulatory Function ◽  
Pcr Analysis ◽  
Biosynthesis Gene Cluster ◽  
Biosynthesis Gene ◽  
Chaetoglobosin A ◽  
Encoding Genes

Gα signaling pathway as well as the global regulator LaeA were demonstrated to positively regulate the biosynthesis of chaetoglobosin A (ChA), a promising biotic pesticide produced by Chaetomium globosum. Recently, the regulatory function of Zn2Cys6 binuclear finger transcription factor CgcheR that lies within the ChA biosynthesis gene cluster has been confirmed. However, CgcheR was not merely a pathway specific regulator. In this study, we showed that the homologs gene of CgcheR (designated as Cgtf1) regulate ChA biosynthesis and sporulation in C. globosum NK102. More importantly, RNA-seq profiling demonstrated that 1,388 genes were significant differentially expressed as Cgtf1 deleted. Among them, a putative C2H2 transcription factor, named Cgtf6, showed the highest gene expression variation in zinc-binding proteins encoding genes as Cgtf1 deleted. qRT-PCR analysis confirmed that expression of Cgtf6 was significantly reduced in CgTF1 null mutants. Whereas, deletion of Cgtf6 resulted in the transcriptional activation and consequent increase in the expression of ChA biosynthesis gene cluster and ChA production in C. globosum. These data suggested that CgTF6 probably acted as an end product feedback effector, and interacted with CgTF1 to maintain a tolerable concentration of ChA for cell survival.

scholarly journals Haemophilus ducreyi Requires the flp Gene Cluster for Microcolony Formation In Vitro

2002 ◽  
Vol 70 (6) ◽  
pp. 2965-2975 ◽  
Author(s):  
Joseph R. Nika ◽  
Jo L. Latimer ◽  
Christine K. Ward ◽  
Robert J. Blick ◽  
Nikki J. Wagner ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Rabbit Model ◽  
Etiologic Agent ◽  
Open Reading Frames ◽  
Haemophilus Ducreyi ◽  
Periodontal Pathogen ◽  
Pcr Analysis ◽  
Phenotypic Trait ◽  
Human Foreskin Fibroblasts

ABSTRACT Haemophilus ducreyi, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in H. ducreyi that encoded predicted protein products with significant homology to those encoded by the tad (for tight adhesion) locus in Actinobacillus actinomycetemcomitans that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this H. ducreyi gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the tad locus; this 15-gene cluster in H. ducreyi was designated flp. RT-PCR analysis indicated that the H. ducreyi flp gene cluster was likely to be a polycistronic operon. Mutations within the flp gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An H. ducreyi mutant with an inactivated tadA gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another H. ducreyi mutant with inactivated flp-1 and flp-2 genes was as virulent as the wild-type parent strain. These results indicate that the flp gene cluster is essential for microcolony formation by H. ducreyi, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.

scholarly journals Structural Analysis and Biosynthesis Gene Cluster of an Antigenic Glycopeptidolipid from Mycobacterium intracellulare

2008 ◽  
Vol 190 (10) ◽  
pp. 3613-3621 ◽  
Author(s):  
Nagatoshi Fujiwara ◽  
Noboru Nakata ◽  
Takashi Naka ◽  
Ikuya Yano ◽  
Matsumi Doe ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Fatty Acyl ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Specific Gene ◽  
Oligosaccharide Structure ◽  
Biosynthesis Gene Cluster ◽  
Mycobacterium Intracellulare ◽  
Serotype 1 ◽  
Species Specific

ABSTRACT Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2′-methyl-3′-hydroxy-4′-methoxy-pentanoyl-amido-3,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-6-deoxy-α-l-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.

scholarly journals Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

1999 ◽  
Vol 181 (1) ◽  
pp. 338-340 ◽  
Author(s):  
Richard van Kranenburg ◽  
Iris I. van Swam ◽  
Joey D. Marugg ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Substrate Specificities ◽  
Exopolysaccharide Biosynthesis

ABSTRACT We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.

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