Genome Sequence of Bacillus megaterium O1, a Saponin-Degrading Bacterium.

Bacillus megaterium strain O1 was isolated from a soapnut (Sapindus saponaria) surface and degrades Quillaja saponin as a sole carbon source. We report the draft genome sequence of B. megaterium O1, which has an estimated size of 5.1 Mb. Study of this isolate will provide insight into mechanisms of saponin degradation.

Genome Sequence of Pseudomonas putida Strain ASAD, an Acetylsalicylic Acid-Degrading Bacterium.

Pseudomonas putida strain ASAD was isolated from compost because of its ability to utilize aspirin (acetylsalicylic acid) as a carbon and energy source. We report the draft genome sequence of strain ASAD, with an estimated length of 6.9 Mb. Study of this isolate will provide insight into the aspirin biodegradation pathway.

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen.

Production of hydrogen gas from light and the inorganic electron donor thiosulfate by Rhodopseudomonas palustris.

A challenge for photobiological production of hydrogen gas (H(2)) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H(2) production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H(2) when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H(2). H(2) production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H(2) production. The possibility to use thiosulfate expands the range of electron-donating compounds for H(2) production by PNSBs beyond biomass-based electron donors.

A new class of homoserine lactone quorum-sensing signals.

Quorum sensing is a term used to describe cell-to-cell communication that allows cell-density-dependent gene expression. Many bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum-sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. Here we show that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium sp. and Silicibacter pomeroyi. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signalling.

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1.

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1.

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.

The acid stress response of the cyanobacterium Synechocystissp. strain PCC 6308.

The cyanobacterium Synechocystissp. strain PCC 6308 has been shown to exhibit predictable physiological responses to acid stress. Originally isolated from a Wisconsin lake, this cyanobacterium grows optimally under alkaline conditions in the laboratory. After acid stress at a pH of between 4.4 and 7.7, cells return to exponential growth following a lag phase. The organism's response to this tolerable acid stress involves cell concentration-dependent neutralization of the external medium to pH 6 or above within 5 min, maintenance of a transmembrane pH gradient, and maintenance of photosystem II efficiency. Lethal acid stress, at a pH below 4.4, results in the formation of aggregates of denatured proteins observed as granules near the cell periphery, the disruption of the transmembrane pH gradient, cell color change to blue, and damage to photosystem II.