Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Development of a shuttle vector for Moraxella catarrhalis

Efforts to perform genetic analysis in Moraxella catarrhalis have been hampered by the lack of a cloning vector. M. catarrhalis strain E22 was previously shown to contain plasmid pLQ510 which lacked a selectable antibiotic resistance marker. Several methods were used to eliminate unnecessary DNA from pLQ510. Then, a 1.2 kb spectinomycin resistance cartridge, a multiple cloning site, and the origin of replication from pACYC184 were cloned into this plasmid backbone to obtain the 7.2 kb plasmid pWW102B. This new plasmid could replicate in M. catarrhalis as well as in both Escherichia coli and Haemophilus influenzae. This shuttle vector was used to clone and express two different M. catarrhalis genes, respectively, encoding an adhesin and a protein involved in serum resistance. When these two plasmids were introduced into appropriate M. catarrhalis mutants, they complemented the phenotypic deficiency of each mutant. This is the first report of functional complementation in trans in this pathogen.     

Three highly conserved proteins catalyze the conversion of UDP-N-acetyl-D-glucosamine to precursors for the biosynthesis of O antigen in Pseudomonas aeruginosa O11 and capsule in Staphylococcus aureus type 5. Implications for the UDP-N-acetyl-L-fucosamine biosynthetic pathway

N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.     

Staphylococcus aureus cap5P Encodes a UDP-N-Acetylglucosamine 2-Epimerase with Functional Redundancy

The serotype 5 capsule gene cluster of Staphylococcus aureus comprises 16 genes (cap5A through cap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that the N-acetylmannosaminuronic acid (ManNAcA) component of the S. aureus serotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded by S. aureus cap5P. Purified Cap5P converted approximately 10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression in S. aureus was unaffected by insertional inactivation of cap5P. Sequence homology searches of the public S. aureus genomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on the S. aureus chromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function in S. aureus was demonstrated by cloning the cap5P homologue from strain Newman and complementing an Escherichia coli rffE mutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of the S. aureus cap5P gene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.     

Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (→ 4)-3-O-Ac-β- D -ManNAcA p -(1 → 4)-α- L -FucNAc p -(1 → 3)-β- D -FucNAc p -(1→). Tn 918 mutagenesis of strain Reynolds yielded a mutant that produced wild-type levels of O-deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn 918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL-LacA-CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S . aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O-deacetylated mutant. When cap5H was provided to the mutant in trans , it fully restored CP5 O-acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.     

Capsular serotype of Staphylococcus aureus in the era of community-acquired MRSA

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.     

Identification of an additional ferric-siderophore uptake gene clustered with receptor, biosynthesis, and fur-like regulatory genes in fluorescent Pseudomonas sp. strain M114.

Five cosmid clones with insert sizes averaging 22.6 kilobases (kb) were isolated after complementation of 22 Tn5-induced Sid- mutants of Pseudomonas sp. strain M114. One of these plasmids (pMS639) was also shown to encode ferric-siderophore receptor and dissociation functions. The receptor gene was located on this plasmid since introduction of the plasmid into three wild-type fluorescent pseudomonads enabled them to utilize the ferric-siderophore from strain M114. The presence of an extra iron-regulated protein in the outer membrane profile of one of these strains was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ferric-siderophore dissociation gene was attributed to pMS639 since it complemented the ferric-siderophore uptake mutation in strain M114FR2. This mutant was not defective in the outer membrane receptor for ferric-siderophore but apparently accumulated ferric-siderophore internally. Since ferric-citrate alleviated the iron stress of the mutant, there was no defect in iron metabolism subsequent to release of iron from the ferric-siderophore complex. Consequently, this mutant was defective in ferric-siderophore dissociation. A fur-like regulatory gene also present on pMS639 was subcloned to a 7.0-kb BglII insert of pCUP5 and was located approximately 7.3 kb from the receptor region. These results established that the 27.2-kb insert of pMS639 encoded at least two siderophore biosynthesis genes, ferric-siderophore receptor and dissociation genes, and a fur-like regulatory gene from the biocontrol fluorescent Pseudomonas sp. strain M114.     

Identification of an additional ferric-siderophore uptake gene clustered with receptor, biosynthesis, and fur-like regulatory genes in fluorescent Pseudomonas sp. strain M114

Five cosmid clones with insert sizes averaging 22.6 kilobases (kb) were isolated after complementation of 22 Tn5-induced Sid- mutants of Pseudomonas sp. strain M114. One of these plasmids (pMS639) was also shown to encode ferric-siderophore receptor and dissociation functions. The receptor gene was located on this plasmid since introduction of the plasmid into three wild-type fluorescent pseudomonads enabled them to utilize the ferric-siderophore from strain M114. The presence of an extra iron-regulated protein in the outer membrane profile of one of these strains was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ferric-siderophore dissociation gene was attributed to pMS639 since it complemented the ferric-siderophore uptake mutation in strain M114FR2. This mutant was not defective in the outer membrane receptor for ferric-siderophore but apparently accumulated ferric-siderophore internally. Since ferric-citrate alleviated the iron stress of the mutant, there was no defect in iron metabolism subsequent to release of iron from the ferric-siderophore complex. Consequently, this mutant was defective in ferric-siderophore dissociation. A fur-like regulatory gene also present on pMS639 was subcloned to a 7.0-kb BglII insert of pCUP5 and was located approximately 7.3 kb from the receptor region. These results established that the 27.2-kb insert of pMS639 encoded at least two siderophore biosynthesis genes, ferric-siderophore receptor and dissociation genes, and a fur-like regulatory gene from the biocontrol fluorescent Pseudomonas sp. strain M114.     

Azide resistance in Rhizobium ciceri linked with superior symbiotic nitrogen fixation

Isolated azide resistant (AzR) native R. ciceri strain 18-7 was resistant to sodium azide at 10 microg/ml. To find if nif-reiteration is responsible for azide resistance and linked to superior symbiotic nitrogen fixation, transposon (Tn5) induced azide sensitive mutants were generated. Using 4 kb nif-reiterated Sinorhizobium meliloti DNA, a clone C4 that complemented azide sensitivity was isolated by DNA hybridization from genomic library of chickpea Rhizobium strain Rcd301. EcoRI restriction mapping revealed the presence of 7 recognition sites with a total insert size of 19.17 kb. Restriction analysis of C4 clone and nif-reiterated DNA (pRK 290.7) with EcoRI and XhoI revealed similar banding pattern. Wild type strain 18-7, mutant M126 and complemented mutant M126(C4) were characterized for symbiotic properties (viz., acetylene reduction assay, total nitrogen content, nodule number and fresh and dry weight of the infected plants) and explanta nitrogenase activity. Our results suggested that azide resistance, nif-reiteration, and superior symbiotic effectiveness were interlinked with no correlation between ex-planta nitrogenase activity and azide resistance in R. ciceri.     

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.     

Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphylococcus aureus

The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.     

Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome.

The complete nucleotide sequence of the cap3A gene of Streptococcus pneumoniae, which is directly responsible for the transformation of some unencapsulated, serotype 3 mutants to the encapsulated phenotype, has been determined. This gene encodes a protein of 394 amino acids with a predicted M(r) of 44,646. Twelve independent cap3A mutations have been mapped by genetic transformation, and three of them have been sequenced. Sequence comparisons revealed that cap3A was very similar (74.4%) to the hasB gene of Streptococcus pyogenes, which encodes a UDP-glucose dehydrogenase (UDP-GlcDH) that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, the donor substances in the pneumococcal type 3 capsular polysaccharide. Furthermore, a PCR-generated cap3A+ gene restored encapsulation in our cap3A mutants as well as in a mutant previously characterized as deficient in UDP-GlcDH (R. Austrian, H. P. Bernheimer, E.E.B. Smith, and G.T. Mills, J. Exp. Med. 110:585-602, 1959). These results support the conclusion that cap3A codes for UDP-GlcDH. We have also identified a region upstream of cap3A that should contain common genes necessary for the production of capsule of any type. Pulsed-field gel electrophoresis and Southern blotting showed that the capsular genes specific for serotype 3 are located near the genes encoding PBP 2X and PBP 1A in the S. pneumoniae chromosome, whereas copies of the common genes (or part of them) appear to be present in different locations in the genome.     

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.