Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

Constitutive expression of erythromycin resistance mediated by the ermAM determinant of plasmid pAM beta 1 results from deletion of 5' leader peptide sequences

We have sequenced the erythromycin resistance determinant (erm) of the Streptococcus faecalis plasmid pAM beta 1 to investigate its relationship to other known resistance determinants. We show that this determinant is strongly (99%) homologous at the DNA level to that of plasmid pAM77 (Streptococcus sanguis) and of transposon Tn917 (S. faecalis). Moreover, nucleotide sequence comparison with the determinants of pAM77 and Tn917 shows that most of the probable regulatory region is absent, providing an explanation for the constitutive expression of the pAM beta 1 erm determinant.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.     

Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.     

粪产碱菌的Tn5转座诱变及吲哚乙酸生物合成特性的研究

粪产碱菌(Alcaligenes faecalis)A1501的吲哚乙酸(IAA)合成需要外源色氨酸参与。在不含色氨酸的限制性培养基中,A1501能良好生长,但不能合成IAA,表明在A1501中存在一条依赖于色氨酸的IAA合成途径。A1501的IAA合成具有菌体密度依赖特性。采用Tn5转座诱变技术构建A1501的突变库,从3500多株Tn5转染子中分离到一株色氨酸营养缺陷型突变株AT63。该Tn5突变株在不含色氨酸的限制性培养基上不能生长,但仍能进行IAA的生物合成,每毫升菌体密度等于10的突变株菌体的IAA合成量为224μg。对突变株AT63的研究表明在A1501中至少存在两条IAA合成途径：一条以色氨酸为合成前体,另一条以吲哚-3-磷酸甘油为前体。Southern杂交结果表明突变株中Tn5插入位点可能位于编码色氨酸合成酶基因上。     

Esp-independent biofilm formation by Enterococcus faecalis

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.     

利用转座子Tn917构建单核细胞增生李斯特菌菌膜形成突变株

单核细胞增生李斯特菌菌膜形成相关基因和调控因子的分离和鉴定是阐明其菌膜形成分子机理的基础。利用原生质体转化这一方式,将带有转座子Tn917的质粒pTV1OK成功地转进了单核细胞增生李斯特菌。通过诱导Tn917转座,得到单核细胞增生李斯特菌Tn917插入突变库,转座率为10-7。经96孔细胞培养板筛选发现,菌株LM49形成菌膜能力明显大于野生型。该菌株在细胞培养板中培养４d后形成的紫色圆环的颜色明显深于野生型。用Tn917特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到相应大小的扩增产物,证实该菌株基因组中有Tn917插入。Tn917的插入使菌株LM49的菌膜形成能力增强。     

Bap, a Staphylococcus aureus surface protein involved in biofilm formation

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.     

Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.

Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.