Physical and genetic characterization of deletions in Streptococcus pneumoniae.

Genetic properties of markers may discriminate between deletions and point mutations. We have designed a physical method for a direct characterization of deletions which also gives an estimate of their size.     

Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae.

Analysis of the sequence data obtained from the 5' portion of the Streptococcus 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 designated cps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniae serotypes with probes specific for the cps19aC, cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported for cps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or the cps19fC 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 the cps regions between cpsB and aliA from 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 of cpsC. 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%.     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

Molecular characterization of an autolysin-defective mutant of Streptococcus pneumoniae

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.     

Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris.

Lac+ plasmid DNA from Streptococcus cremoris H2 was subcloned with an Escherichia coli vector on a 3.5-kilobase-pair PstI-AvaI fragment. Genetic analysis of the cloned DNA was possible because linear Lac+ DNA fragments were productive in the S. sanguis transformation system. Complementation of S. sanguis Lac-mutants showed that the 3.5-kilobase-pair fragment included the structural gene for 6-phospho-beta-D-galactosidase and either enzyme II-lac or factor III-lac of the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system. Expression of the S. cremoris-like 40,000-dalton 6-phospho-beta-D-galactosidase in S. sanguis Lac+ transformants, rather than the 52,000-dalton wild-type S. sanguis enzyme, demonstrated the occurrence of gene replacement and not gene repair. The evidence supports chromosomal integration as the mechanism by which S. sanguis Lac- recipients are converted to a Lac+ phenotype after transformation with Lac+ DNA. Southern blot data suggest that the Lac+ DNA does not reside on a transposon, but that integration always occurs within a specific HincII fragment of the recipient chromosome. Hybridization experiments demonstrate homology between the S. cremoris Lac+ DNA and cellular DNA from Lac+ strains of Streptococcus lactis, S. mutans, S. faecalis, and S. sanguis.     

Molecular characterization of the klebicin B plasmid of Klebsiella pneumoniae.

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.     

19群肺炎链球菌国家标准菌株分子特征分析及在菌株质量控制中的应用

目的明确19群肺炎链球菌(Streptococcus pneumoniae)国家标准菌株的分子特征,为完善中国肺炎链球菌国家标准菌株的质量标准提供依据。方法在传统肺炎链球菌菌种检定方法的基础上,应用16S rRNA基因分析、聚合酶链式反应(polymerase chain reaction,PCR)血清分型、多位点序列分型(multilocus sequence type,MLST)和脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型等多种分子生物学质控方法,对来源于中国医学细菌保藏管理中心的20株19群肺炎链球菌国家标准菌株进行分析。结果 20株19群肺炎链球菌的16S rRNA基因序列与肺炎链球菌模式株NCTC 7465的16S rRNA基因序列的相似性在99.64%～100%之间,碱基差异0～5 bp。PCR血清分型结果表明:11株19F型菌株均检测到大小为304 bp的19F型特异性扩增条带,6株19A型菌株均检测到大小为478 bp的19A型特异性扩增条带,PCR血清分型结果与血清凝集试验结果一致。MLST分型结果表明,相同血清型的菌株可以具有不同的序列型(sequence type,ST)。共获得12个ST型,其中6个ST型(10496、10497、10501、10502、10503和10509)为首次报道。PFGE分型结果显示:各型别菌株各具有其特征性PFGE带型(9～17条带),相同血清型或ST型菌株可能具有不同的PFGE带型。共存在18种不同的PFGE带型,其中19F型和19A型菌株中各有一对菌株的ST型和PFGE图谱完全一致。菌株的传代稳定性考察结果显示:在25代以内31708菌株的PFGE带型未发生变化,遗传特征是稳定的。结论 16S rRNA基因分析、PCR血清分型、MLST分型和PFGE分型等分子生物学方法应用于中国肺炎链球菌国家标准菌株的质量控制,可获得更加全面的肺炎链球菌标准菌株身份信息数据(包括序列、PCR扩增片段、序列型、图谱等),为进一步完善中国肺炎链球菌国家标准菌株的质量标准提供依据和数据支撑。     

Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity

The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.     

Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor

The type 3 synthase from Streptococcus pneumoniae is a processive beta-glycosyltransferase that assembles the type 3 polysaccharide [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[(14)C]Glc or UDP-[(14)C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.     

Molecular cloning, expression, and characterization of dnaK in Streptococcus pneumoniae

DnaK is known to be highly conserved in all species and is a major immunogen in Streptococcus pneumoniae. To elucidate the role of dnaK in S. pneumoniae, dnaK was cloned in Escherichia coli using a homologous dnaK probe generated by PCR. The His-tagged DnaK was overexpressed in soluble form and purified from E. coli. Alignment of the deduced DnaK amino acid sequence from nucleotide sequences of the cloned dnaK revealed high homology with DnaK analogs in E. coli (53%) and Staphylococcus aureus (73%). However, anti-pneumococcal DnaK antiserum did not crossreact with DnaK analogs in E. coli, S. aureus and human cells suggesting that pneumococcal DnaK might be a good candidate as a vaccine.     

Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

Molecular characterization of staphyloferrin B biosynthesis in Staphylococcus aureus

Siderophores are iron-scavenging molecules produced by many microbes. In general, they are synthesized using either non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) pathways. Staphylococcus aureus produces siderophores, of which the structures of staphyloferrin A and staphyloferrin B are known. Recently, the NIS biosynthetic pathway for staphyloferrin A was characterized. Here we show that, in S. aureus , the previously identified sbn ( s iderophore b iosy n thesis) locus encodes enzymes required for the synthesis of staphyloferrin B, an α-hydroxycarboxylate siderophore comprised of l -2,3-diaminopropionic acid, citric acid, 1,2-diaminoethane and α-ketoglutaric acid. Staphyloferrin B NIS biosynthesis was recapitulated in vitro , using purified recombinant Sbn enzymes and the component substrates. In vitro synthesized staphyloferrin B readily promoted the growth of iron-starved S. aureus , via the ABC transporter SirABC. The SbnCEF synthetases and a decarboxylase, SbnH, were necessary and sufficient to produce staphyloferrin B in reactions containing component substrates l -2,3-diaminopropionic acid, citric acid and α-ketoglutaric acid. Since 1,2-diaminoethane was not required, this component of the siderophore arises from the SbnH-dependent decarboxylation of a 2,3-diaminoproprionic acid-containing intermediate. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analyses of a series of enzyme reactions identified mass ions corresponding to biosynthetic intermediates, allowing for the first proposed biosynthetic pathway for staphyloferrin B.