The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O-antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O-antigen units, which are subsequently assembled to form polymerized O-antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O-antigen-like structure and that B. pertussis LPS is similar to semi-rough LPS seen in some mutants of enteric bacteria.     

Identification of a plasmid-borne locus in Rhizobium etli KIM5s involved in lipopolysaccharide O-chain biosynthesis and nodulation of Phaseolus vulgaris.

Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgusA30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv(-) Fix(-) phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (alpha-D-GalA-1-->4[alpha-D-GalA-1-->5]-Kdo) and tetrasaccharide (alpha-D-Gal-1-->6[alpha-D-GalA-1-->4]-alpha-D-Man-1-->5Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in trans on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host Phaseolus vulgaris. The mTn5 insertion in KIM-G1 was located at the N terminus of a putative alpha-glycosyltransferase, which most likely had a polar effect on a putative beta-glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the lpsbeta2 gene recently reported for R. etli CFN42.     

Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus.

Tn10 insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::Tn10 insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype.     

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage φR1-37 when grown at 37°C but not when grown at 22°C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22°C. The transposon library of YeO3-c was grown at 37°C and screened for phage φR1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the trs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarity to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus ; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage φR1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk—hemH and galE—gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage φR1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae

Abstract The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant ( aroB ) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens , the HitABC proteins of Haemophilia influenzae , the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica . The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin genes.     

Identification by saturation mutagenesis of a single residue involved in the alpha-galactosidase AgaB regioselectivity

The -galactosidase AgaB of Bacillus stearothermophilus displays a major 1,6 and a minor 1,3 regioselectivity. The wild-type enzyme was subjected to directed evolution (random mutagenesis and in vitro recombination) using a double screening strategy based on the elimination of the 1,6 regioselectivity and the analysis by TLC of the transglycosylation products. One of the AgaB mutants (E500) exhibited a new 1,2 regioselectivity and a rather high level of transglycosylation. The corresponding gene contains 10 mutations compared to the agaB gene and we demonstrated by saturation mutagenesis that the G442R substitution strongly contributes to the emergence of this new regioselectivity. Moreover, other single point mutations at this position led to new mutants displaying other kinds of regioselectivity demonstrating the importance of this position in the subtle kinetic control of transglycosylation.     

Molecular analysis of a complex locus from Haemophilus influenzae invoked in phase-variable lipopolysaccharide biosynthesis

A chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. lic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. ORF2 encodes the UDP-galactose-4-epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. ORF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3' end of ORF1, all of galE, and the 5' end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.     

Identification,cloning and characterization of rfaE of Actinobacillus pleuropneumoniae serotype 1, a gene involved in lipopolysaccharide inner-core biosynthesis

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants (rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP-heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.     

The properties of sequences involved in insertion and deletion mutagenesis in rodents

We analyzed nucleotide sequences that were inserted to or deleted from genomic DNA during the divergence from common ancestor. The median length of these sequences is 3 nucleotides; they are enriched with palindromes and often repeat in adjacent DNA.     

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.     

Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.     

Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.     

The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity

We constructed a set of plasmid-encoded internal deletion mutants within the gene for the adsorption protein (g3p) of phage IKe. All mutant proteins still contain the signal and membrane anchor sequence, as those are known to be indispensable for proper localization and hence assembly of the g3p into phage. These various deletions comprise all internal parts of the protein and are properly incorporated into phage, which remarkably shows that signal and anchor sequence are sufficient for incorporation of g3p. The data furthermore reveal that two separate sections within the IKe g3p are essential for infection: one amino-terminal, preceding the glycine-rich stretch, and the other carboxy-terminal. We conclude that this latter domain is involved in penetration because mutants lacking it are not infectious, but still bind to the receptor. The amino-terminal region, essential for infection, bears the receptor-recognizing domain and a sequence homologous to the penetration domain of the evolutionary related Ff phages, which is probably also involved in penetration of phage IKe. The prominent glycine-rich stretch of the IKe g3p is not essential for infection but significantly promotes it.     

The paramutated SULFUREA locus of tomato is involved in auxin biosynthesis

The tomato (Solanum lycopersicum) sulfurea mutation displays trans-inactivation of wild-type alleles in heterozygous plants, a phenomenon referred to as paramutation. Homozygous mutant plants and paramutated leaf tissue of heterozygous plants show a pigment-deficient phenotype. The molecular basis of this phenotype and the function of the SULFUREA gene (SULF) are unknown. Here, a comprehensive physiological analysis of the sulfurea mutant is reported which suggests a molecular function for the SULFUREA locus. It is found that the sulf mutant is auxin-deficient and that the pigment-deficient phenotype is likely to represent only a secondary consequence of the auxin deficiency. This is most strongly supported by the isolation of a suppressor mutant which shows an auxin overaccumulation phenotype and contains elevated levels of indole-3-acetic acid (IAA). Several lines of evidence point to a role of the SULF gene in tryptophan-independent auxin biosynthesis, a pathway whose biochemistry and enzymology is still completely unknown. Thus, the sulfurea mutant may provide a promising entry point into elucidating the tryptophan-independent pathway of IAA synthesis.     

Cloning and characterization of a gene from Pasteurella haemolytica A1 involved in lipopolysaccharide biosynthesis

Abstract A Pasteurella haemolytica A1 gene involved in the biosynthesis of a moiety on the core of the lipopolysaccharide molecule has been cloned and characterized. Escherichia coli clones which carry this gene showed an alteration of its lipopolysaccharide migration profile on tricine SDS-PAGE and exhibited resistance to the core-specific phage U3. In addition, lipopolysaccharide extracted from the E. coli clones was recognized by an anti-corespecific antiserum, but not by antiserum specific for the O antigen of P. haemolytica A1 lipopolysaccharide. Nucleotide sequence analysis of the cloned DNA identified an open reading frame ( lpsA ) coding for a protein of 263 amino acids which showed significant homology with a Haemophilus influenzae type b lipooligosaccharide biosynthesis gene. PCR amplification of genomic DNA, using primers based on the P. haemolytica A1 lpsA sequence, yielded products from only the A biotypes of P. haemolytica .     

apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

Antibodies to meningococcal lipopolysaccharide in different forms of meningococcal infection

The results of the determination of antibodies to lipopolysaccharide (LPS) in 270 patients with different forms of meningococcal infection and in 816 healthy persons by means of the passive hemagglutination test are presented. The role of antibodies to LPS in the formation of humoral immunity to meningococci in sick children and adults is shown. Different forms of meningococcal infection have been found to have their specific features of the accumulation of antibodies to LPS. As revealed, the time of the sanation of liquor and the level of antibodies to LPS are unrelated, which indicates that antibodies to LPS may play some role in the pathogenesis of meningococcal infection.     

Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.