We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (TnSSI) as a cloning probe. The phase (5, 6). At the same time, the production of many proteins essential for growth and cell division is shut off. The regulation system that governs this changeover can be regarded as a metabolic toggle switch that is set at the end of exponential phase for accessory protein synthesis. When a new growth cycle is initiated (e.g., by dilution into fresh medium), the switch is reset for the synthesis of exponential-phase proteins. Neither the nature of the switch nor the identity of the metabolic factors involved is known. Pleiotropic mutations affecting the production of accessory proteins in S. aureus have been described by several groups (1, 10, 34), and it is likely that their analysis may be informative about this regulation system. Commonly, these mutations block post-exponentialphase synthesis of the following proteins: serine protease, nuclease, lipase, fibrinolysin, a-hemolysin, ,-hemolysin, 8-hemolysin, enterotoxin B, and toxic shock syndrome toxin-1 (TSST-1), whereas production of certain other exoproteins, including protein A and coagulase, is increased (1, 25

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Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5 noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.

Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346 -PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.Most bacteria possess multiple sets of two-component regulatory systems (2CSs), 2 which are used for the reception of and response to environmental challenges (1). Typical 2CSs are composed of a sensor and a response regulator. The sensor is normally a transmembrane histidine kinase that detects a specific environmental stimulus and auto-phosphorylates a conserved histidine residue within its transmitter domain. The phosphoryl group is subsequently transferred from the histidine residue to an aspartic acid on the cognate regulator, which then activates the expression of genes required for countering the environmental stress. In addition to this basic "His 3 Asp" type of phosphotransfer mechanism, more complex multistep phosphorelay 2CSs also exist, in which the sensor harbors two extra domains as follows: a receiver domain containing a phosphor-accepting Asp and a Hpt domain. The most well characterized examples of the complex type 2CS are the anaerobic regulator ArcAB of Escherichia coli (2) and virulence-associated regulator BvgAS of Bordetella spp. (3). Both are capable of performing a multistep His 3 Asp 3 His 3 Asp phosphorelay.An intermediate group of sensors are known as the hybrid sensors. The hybrid-type sensors, which contain a kinase and a receiver domain but lack an Hpt domain, are believed to require another protein to provide the Hpt domain for their signal transduction ...

A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.