scholarly journals Construction and Characterization of an Acapsular Mutant of Mannheimia haemolytica A1

2002 ◽  
Vol 70 (5) ◽  
pp. 2622-2629 ◽  
Author(s):  
Linda J. McKerral ◽  
Reggie Y. C. Lo
Keyword(s):  
Southern Hybridization ◽  
Capsular Polysaccharide ◽  
Calf Serum ◽  
Mannheimia Haemolytica ◽  
Knockout Mutant ◽  
Chloramphenicol Resistance ◽  
Polysaccharide Biosynthesis ◽  
Dna Fragment ◽  
Microscopy Examination

ABSTRACT The nmaA and nmaB genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in Mannheimia haemolytica A1. A chloramphenicol resistance (Cmr) cassette cloned behind an M. haemolytica A1 promoter, plpcat, was created and used to interrupt nmaA and nmaB. A 1.3-kbp DNA fragment that encompasses part of nmaA and nmaB was replaced by the 1.0-kbp plpcat, resulting in a knockout mutant which is Cmr and unable to synthesize N-acetylmannosamine (ManNAc) and N-acetylmannosaminuronic acid (ManNAcA). The DNA replacement was confirmed by Southern hybridization and PCR analyses of the nmaA and nmaB loci. Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain. The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum. Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.

scholarly journals The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Nehmé SAKSOUK ◽  
Ludovic PELOSI ◽  
Pierre COLIN-MOREL ◽  
Manel BOUMEDIENNE ◽  
Patricia L. ABDIAN ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Biochemical Characterization ◽  
Repeat Unit ◽  
Sugar Residue ◽  
Functional Identification ◽  
Polysaccharide Biosynthesis ◽  
Sugar Addition ◽  
Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

scholarly journals Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides

2009 ◽  
Vol 191 (6) ◽  
pp. 1891-1900 ◽  
Author(s):  
Jinghua Yang ◽  
Mary Ritchey ◽  
Yasuo Yoshida ◽  
C. Allen Bush ◽  
John O. Cisar
Keyword(s):  
Dental Plaque ◽  
Molecular Basis ◽  
Capsular Polysaccharide ◽  
Polysaccharide Biosynthesis ◽  
Streptococcus Oralis ◽  
High Resolution Nmr ◽  
Close Relationship ◽  
Plaque Biofilm ◽  
Biofilm Community

ABSTRACT The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2ribitol-5-PO4 −-6Galfβ1-3Galβ1]n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

Characterization of the rrnB Operon of the Plant Pathogen Rhodococcus fascians and Targeted Integrations of Exogenous Genes at rrn Loci

1998 ◽  
Vol 64 (4) ◽  
pp. 1276-1282 ◽  
Author(s):  
Agustín Pisabarro ◽  
António Correia ◽  
Juan F. Martín
Keyword(s):  
Virulent Strain ◽  
Normal Growth ◽  
23S Rrna ◽  
Chloramphenicol Resistance ◽  
Gram Positive Bacteria ◽  
Integrative Vector ◽  
Rhodococcus Fascians ◽  
Dna Fragment ◽  
Exogenous Genes

ABSTRACT A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5′ and 3′ ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with therrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strainR. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as R. fascians, produced fiverrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

Duplication of insertion element IS50 associated with Tn5 transposition in Azospirillum brasilense

1998 ◽  
Vol 44 (11) ◽  
pp. 1110-1113 ◽  
Author(s):  
Anil K Tripathi ◽  
Rachna Tripathi ◽  
Abhijit Ganguli ◽  
Marco Bazzicalupo
Keyword(s):  
Homologous Recombination ◽  
Restriction Endonuclease ◽  
Azospirillum Brasilense ◽  
Southern Hybridization ◽  
Recombinant Dna ◽  
Nucleotide Sequencing ◽  
Insertion Element ◽  
Dna Fragment ◽  
Endonuclease Mapping

The characterization of a DNA fragment with a Tn5 insertion in a regulatory nif gene of Azospirillum brasilense is reported. Restriction endonuclease mapping, Southern hybridization with a Tn5 probe, and nucleotide sequencing revealed that IS50 had duplicated in Tn5. The duplication of an IS50 element suggests the occurrence of a replicative mechanism of transposition. A strategy, based on the bacterial ability of homologous recombination that was used to precisely eliminate Tn5 along with the duplicated IS50 element, is presented.Key words: Tn5, IS50, Azospirillum brasilense, recombinant DNA.

scholarly journals Interstrain Variation of the Polysaccharide B Biosynthesis Locus of Bacteroides fragilis: Characterization of the Region from Strain 638R

1999 ◽  
Vol 181 (19) ◽  
pp. 6192-6196 ◽  
Author(s):  
Laurie E. Comstock ◽  
Michael J. Coyne ◽  
Arthur O. Tzianabos ◽  
Dennis L. Kasper
Keyword(s):  
Capsular Polysaccharide ◽  
Bacteroides Fragilis ◽  
Polysaccharide Biosynthesis

ABSTRACT The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

scholarly journals Analysis of the 5′ Portion of the Type 19A Capsule Locus Identifies Two Classes of cpsC, cpsD, andcpsE Genes in Streptococcus pneumoniae

1999 ◽  
Vol 181 (11) ◽  
pp. 3599-3605 ◽  
Author(s):  
Judy K. Morona ◽  
Renato Morona ◽  
James C. Paton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Southern Hybridization ◽  
Capsular Polysaccharide ◽  
Sequence Data ◽  
Direct Sequencing ◽  
Southern Hybridization Analysis ◽  
Polysaccharide Biosynthesis ◽  
Capsule Locus ◽  
Pcr Products ◽  
Long Range Pcr

ABSTRACT Analysis of the sequence data obtained from the 5′ portion of theStreptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designatedcps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniaeserotypes with probes specific for the cps19aC,cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported forcps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or thecps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify thecps regions between cpsB and aliAfrom a variety of pneumococcal serotypes. Direct sequencing of the 5′ end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes ofcpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.

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