We performed a microbial community analysis of biofilms inhabiting thermal (35 to 50 degrees C) waters more than 60 m below the ground surface near Acquasanta Terme, Italy. The groundwater hosting the biofilms has 400 to 830 microM sulfide, <10 microM O(2), pH of 6.3 to 6.7, and specific conductivity of 8,500 to 10,500 microS/cm. Based on the results of 16S rRNA gene cloning and fluorescent in situ hybridization (FISH), the biofilms have low species richness, and lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms have <90% 16S rRNA similarity to their closest relatives in public databases and may represent novel sulfate-reducing bacteria. The Acquasanta biofilms share few species in common with Frasassi cave biofilms (13 degrees C, 80 km distant) but have a similar community structure, with representatives in the same major clades. The ecological success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is consistent with previous observations of their dominance in sulfidic cave waters with turbulent water flow and high dissolved sulfide/oxygen ratios.

ABSTRACT The saePQRS system of Staphylococcus aureus controls the expression of major virulence factors and encodes a histidine kinase (SaeS), a response regulator (SaeR), a membrane protein (SaeQ), and a lipoprotein (SaeP). The widely used strain Newman is characterized by a single amino acid change in the sensory domain of SaeS (Pro18 in strain Newman [SaeS P ], compared with Leu18 in other strains [SaeS L ]). SaeS P determines activation of the class I sae target genes ( coa , fnbA , eap , sib , efb , fib , sae ), which are highly expressed in strain Newman. In contrast, class II target genes ( hla , hlb , cap ) are not sensitive to the SaeS polymorphism. The SaeS L allele ( saeS L ) is dominant over the SaeS P allele, as shown by single-copy integration of saePQRS L in strain Newman, which results in severe repression of class I target genes. The differential effect on target gene expression is explained by different requirements for SaeR phosphorylation. From an analysis of saeS deletion strains and strains with mutated SaeR phosphorylation sites, we concluded that a high level of SaeR phosphorylation is required for activation of class I target genes. However, a low level of SaeR phosphorylation, which can occur independent of SaeS, is sufficient to activate class II target genes. Using inducible saeRS constructs, we showed that the expression of both types of target genes is independent of the saeRS dosage and that the typical growth phase-dependent gene expression pattern is not driven by SaeRS.

ABSTRACT Staphylococcus aureus reacts to changing environmental conditions such as heat, pH, and chemicals through global regulators such as the sae ( S. aureus exoprotein expression) two-component signaling system. Subinhibitory concentrations of some antibiotics were shown to increase virulence factor expression. Here, we investigated the S. aureus stress response to sublethal concentrations of a commonly used biocide (Perform), by real-time quantitative PCR (qRT-PCR), promoter activity assay, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a flow cytometric invasion assay. Perform, acting through the production of reactive oxygen species, generally downregulated expression of extracellular proteins in strains 6850, COL, ISP479C but upregulated these proteins in strain Newman. Upregulated proteins were sae dependent. The Perform component SDS, but not paraquat (another oxygen donor), mimicked the biocide effect. Eap (extracellular adherence protein) was most prominently augmented. Upregulation of eap and sae was confirmed by qRT-PCR. Promoter activity of sae P1 was increased by Perform and SDS. Both substances enhanced cellular invasiveness, by 2.5-fold and 3.2-fold, respectively. Increased invasiveness was dependent on Eap and the sae system, whereas agr , sarA , sigB , and fibronectin-binding proteins had no major effect in strain Newman. This unique response pattern was due to a point mutation in SaeS (the sensor histidine kinase), as demonstrated by allele swapping. Newman saePQRS ISP479C behaved like ISP479C, whereas saePQRS Newman rendered ISP479C equally responsive as Newman. Taken together, the findings indicate that a point mutation in SaeS of strain Newman was responsible for increased expression of Eap upon exposure to sublethal Perform and SDS concentrations, leading to increased Eap-dependent cellular invasiveness. This may be important for understanding the regulation of virulence in S. aureus .

ABSTRACT The two-component system SaeRS of Staphylococcus aureus is closely involved in the regulation of major virulence factors. However, little is known about the signals leading to saeRS activation. A total of four overlapping transcripts (T1 to T4) from three different transcription starting points are expressed in the sae operon. We used a β-galactosidase reporter assay to characterize the putative promoter regions within the saeRS upstream region. The main transcript T2 is probably generated by endoribonucleolytic processing of the T1 transcript. Only two distinct promoter elements (P1 and P3) could be detected within the saeRS upstream region. The P3 promoter, upstream of saeRS, generates the T3 transcript, includes a cis-acting enhancer element and is repressed by saeRS. The most distal P1 promoter is strongly autoregulated, activated by agr, and repressed by sigma factor B. In strain Newman a mutation within the histidine kinase SaeS leads to a constitutively activated sae system. Evaluation of different external signals revealed that the P1 promoter in strain ISP479R and strain UAMS-1 is inhibited by low pH and high NaCl concentrations but activated by hydrogen peroxide. The most prominent induction of P1 was observed at subinhibitory concentrations of α-defensins in various S. aureus strains, with the exception of strain ISP479R and strain COL. P1 was not activated by the antimicrobial peptides LL37 and daptomycin. In summary, the results indicate that the sensor molecule SaeS is activated by alteration within the membrane allowing the pathogen to react to phagocytosis related effector molecules.

Deep inside the Frasassi cave complex in the foreland fold and thrust belt of the northeastern Apennines (central Italy), the remains of hundreds of eels (Anguilla anguilla) are found scattered on the shores of phreatic lakes up to 5 m above the water table. These sub-fossil eels, and the speleothemic calcite encrusting some of them, offer the rare opportunity for radiocarbon dating leading to a geochronologic scale for the shorelines, which record the lowering of the water table and the uplifting of the Frasassi area through the Holocene. The lakes' margins are contoured by white microcrystal line calcite rinds, which also record the progressive lowering of the water table. Detailed surveying revealed that these rinds are no longer horizontal, but slightly tilted toward ENE. Thus these rinds record a recent history not only of uplifting, but also of tectonic tilting of this region. The results of our analyses indicate that the Apennine area around Frasassi has been rising, for the past 8000 years, at a mean rate of 0.6 mm/yr, which is consistent with uplifting rates estimated from the step topography arrangement of interglacial fluvial terraces for the whole Quaternary period in this region. This work demonstrates how an interdisciplinary approach to speleologic research can provide a significant contribution to active tectonic studies. (c) 2007 Elsevier B.V. All rights reserved.