Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system

The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.     

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.     

Scooter, a new active transposon in Schizophyllum commune, has disrupted two genes regulating signal transduction

Two copies of scooter, a DNA-mediated transposon in the basidiomycetous fungus Schizophyllum commune, were characterized. Scooter is the first transposon isolated from S. commune. Scooter creates 8-bp target site duplications, comparable to members of the hAT superfamily, and has 32-bp terminal inverted repeats. Both copies of scooter are nonautonomous elements capable of movement. Southern blot hybridizations show that scooter-related sequences are present in all S. commune strains tested. Scooter-1 was identified initially as an insertion in the Bbeta2 pheromone receptor gene, bbr2, leading to a partial defect in mating. Scooter-2 spontaneously disrupted a gene to produce the frequently occurring morphological mutant phenotype known as thin. The scooter-2 insert permitted cloning of the disrupted gene, thn1, which encodes a putative regulator of G protein signaling (RGS) protein. Spontaneous insertion of scooter into genes with identifiable mutant phenotypes constitutes the first evidence of active transposition of a DNA-mediated transposon in a basidiomycete.     

Phosphodiesterase activity of CvfA is required for virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.     

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.     

A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.     

Cadmium-regulated gene fusions in Pseudomonas fluorescens

To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.     

Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.     

Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus

Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.     

Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export.

To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions. We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking). Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation. Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p. Temperature-sensitive alleles of DBP5/RAT8 were prepared. In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature. Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region. Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs). The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.     

Generating mutant rats using the Sleeping Beauty transposon system

The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3′-rapid amplification of cDNA ends (3′RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.