Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

Summary A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.     

Isolation and characterization of unsaturated fatty acid auxotrophs of Streptococcus pneumoniae and Streptococcus mutans

Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.     

Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae.

Several pneumococcal phages showing a morphology completely different from those of all other previously found pneumococcal bacteriophages have been isolated. Bacteriophage Cp-1, one of the phages isolated, showed an irregular hexagonal structure and a short tail of 20 nm. The virion density was 1.46 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of nine polypeptides. The polypeptide showing a molecular weight of 39,000 accounted for more than the 90% of the total protein. The nucleic acid of Cp-1 was linear, double-stranded DNA with a mean length of 6.3 microns and a guanine-plus-cytosine content of 41%; its buoyant density was 1.699 and 1.422 g/cm3 in CsCl and CS2SO4, respectively. Its sedimentation coefficient (S20,w) was 19S. Cp-1 DNA showed a remarkable resistance to a large number of restriction endonucleases. A total of 12 fragments, ranging in molecular weight from 1.3 X 10(6) to 0.09 X 10(6), were produced by AluI, two fragments (molecular weight, 5.5 X 10(6) and 0.9 X 10(6)) were generated by HindIII, and two fragments (molecular weight, 6.0 X 10(6) and 5.7 X 10(6)) were produced by HaeIII. The easy visualization of th plaques produced by Cp-1, the small size of Cp-1 DNA (12 X 10(6) daltons), and other biological and physiochemical properties make this phage potentially useful for genetic studies.     

Isolation and characterization of an uncoupler-resistant mutant of Methanothermobacter thermautotrophicus

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the protonophorous uncoupler TCS was isolated. The mutant strain exhibited increased CH(4) formation and elevated level of ATPase activity under non-growing conditions. ATP synthesis driven by methanogenic electron transport as well as by potassium diffusion potential in the presence of either H(+) or Na(+) ions was markedly diminished in the mutant strain. An abundant membrane-associated protein complex with molecular mass approximately 670 kDa was detected in the mutant strain after native PAGE. The results indicate that TCS resistance in this mutant has arisen as a consequence of mutation(s) that affects a specific locus coding for an uncoupler binding protein(s) and/or modulate the activity of unidentified ATPase.     

Purification and properties of Streptococcus pneumoniae neuraminidase

Considerable amounts (200 units/ml) of neuraminidase activity were detected in middle ear effusion of children (age 1 month-10 years) and its presence was highly correlated with the presence of Streptococcus pneumoniae. When isolates of this organism are cultured, neuraminidase activity appears in the growth medium during the exponential phase of growth. In order to study the role of this enzyme in the pathology of otitis media we have developed a method for its purification. The enzyme was purified over 5,800-fold by removing the organism and passing the culture broth through a series of affinity and ion-exchange columns. The overall yield was 2 mg enzyme protein and the final specific activity was 1.8 X 10(6) units/mg protein. A molecular weight of 65,000 was estimated by SDS-PAGE and gel filtration chromatography. The Stokes radius of neuraminidase was calculated to be 32 A, its isoelectric point was 7.2, and its pH optimum was 6.0. In terms of specificity, the enzyme catalyzed the hydrolysis of sialic acid linkages in mucin, glycoproteins, and gangliosides: bovine submaxillary mucin supported the highest catalytic efficiency, and alpha-1-antitrypsin the lowest. Neuraminidase acted on at least three linkage classes of substrates, alpha-2,6 and alpha-2,3 linkages of N-acetylneuraminic acid to galactose, and alpha-2,6 linkages to N-acetyl-galactosamine.     

Isolation and characterization of an aminolevulinate-requiring Rhodobacter capsulatus mutant.

Using transposon Tn5 mutagenesis, we isolated a mutant strain of Rhodobacter capsulatus that requires aminolevulinate for growth. Southern blot analysis indicated that this strain has a single Tn5 insertion. The addition of 0.1 mM aminolevulinate to the medium allowed the mutant to grow either aerobically or photosynthetically with generation times similar to those of the parental strain. When grown photosynthetically, bacteriochlorophyll accumulation increased with increasing aminolevulinate concentration. The mutant strain had only 10% of the normal aminolevulinate synthase activity, but it had a normal level of porphobilinogen synthase activity. The requirement for aminolevulinate could be satisfied by porphobilinogen, hemin, or protoporphyrin. While the mutant grew well on agar plates containing any of these substrates, growth in liquid media containing hemin or protoporphyrin was poor. Introduction of an R' factor containing all the known R. capsulatus bch genes into the mutant strain did not relieve the requirement for aminolevulinate, suggesting that the Tn5 insertion is not within the bch region.     

Phenotypic characterization of Streptococcus pneumoniae biofilm development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.     

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.     

Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.     

Isolation,identification of Streptococcus pneumoniae from infected rhesus monkeys and control efficacy

Background Streptococcus pneumoniae can cause a wide variety of illnesses. Primate animals can be infected by the pneumococcus. A disease occurred among rhesus monkeys in winter 2006. Methods Routine clinical observation, necropsies, bacteriological examinations were conducted, and PCR, pathogenicity to BALB/c mice and antibiotic susceptibility test were examined additionally. Results We conclude that the agent is S. pneumoniae. Based on the antibiotic susceptibility test, a dose of 20 mg/kg body weight daily of Erythromycin was given intramuscular injection for 5 days, resulting in the disappearance of clinical signs, and no newly case reappear be observed till today. Conclusions Therefore, it is suggested that the outbreak of respiratory disease in the rhesus monkeys was because of transmission of S. pneumoniae among rhesus monkeys. The antibiotic therapy finding underscores the utility of Erythromycin to cure the infected rhesus monkeys without causing side effects and without contributing to the further development of antibiotic resistance.     

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.     

Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.     

Isolation and characterization of an actinomycin D-sensitive mutant of Saccharomyces cerevisiae.

A single mutation in Saccharomyces cerevisiae conferred sensitivity to low concentrations of actinomycin D. Treatment with actinomycin D preferentially inhibited synthesis of rRNA's. Residual rRNA synthesized was processed normally. Total protein synthesis and inducibility of the enzyme maltase were relatively unaffected at concentrations of actinomycin D which severely inhibited rRNA synthesis.     

Isolation and partial characterization of an argR mutant of Salmonella typhimurium.

An arginine regulatory mutant (i.e., mutated in the argR gene) has been isolated from a strain of Salmonella typhimurium LT2. The argR mutant was found to excrete arginine into the growth medium with glycerol but not glucose as carbon source. Constitutive synthesis of arginine biosynthetic enzymes was observed. Whereas previous results (A. T. Abd-E1-A1 and J. L. Ingraham, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K169, p. 175) have shown constitutive synthesis of carbamyl phosphate synthetase in the argR mutant, the regulation of the synthesis of the last five enzymes of the pyrimidine pathway was unaffected. However, in pyrH mutants, known to exhibit derepressed synthesis of the pyrimidine enzymes, a 10-fold derepression of ornithine transcarbamylase was observed.