Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux of glycine through several pathways, including the metabolism of glycine to produce other amino acids, entry into the trichloroacetic acid cycle, and the production of virulence factors such as hydrogen cyanide. In this study, we characterized GcsR, a transcriptional regulator that activates the expression of genes involved in P. aeruginosa PAO1 glycine metabolism. Our work reveals that GcsR is the founding member of a novel class of TyrR-like EBPs that likely regulate glycine metabolism in Pseudomonadales.

Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eateutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and ␤-galactosidase fusions, the enhancer-binding protein PA4021 and the sigma factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCEThe results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the sigma factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings. E thanolamine serves as a source of carbon and nitrogen for a variety of bacteria, including members of the Enterobacteriaceae, Pseudomonadaceae, and Firmicutes (1-5). From studies mostly centered on Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli, there is now a basic understanding of the catabolic steps involved in ethanolamine utilization. Extracellular ethanolamine enters the bacterial cell through simple diffusion or carrier-me...

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bWhen starved for nutrients, Myxococcus xanthus produces a biofilm that contains a mat of rod-shaped cells, known as peripheral rods, and aerial structures called fruiting bodies, which house thousands of dormant and stress-resistant spherical spores. Because rod-shaped cells differentiate into spherical, stress-resistant spores and spore differentiation occurs only in nascent fruiting bodies, many genes and multiple levels of regulation are required. Over the past 2 decades, many regulators of the temporal and spatial expression of M. xanthus sporulation genes have been uncovered. Of these sporulation gene regulators, twocomponent signal transduction circuits, which typically contain a histidine kinase sensor protein and a transcriptional regulator known as response regulator, are among the best characterized. In this review, we discuss prototypical two-component systems (Nla6S/Nla6 and Nla28S/Nla28) that regulate an early, preaggregation phase of sporulation gene expression during fruiting body development. We also discuss orphan response regulators (ActB and FruA) that regulate a later phase of sporulation gene expression, which begins during the aggregation stage of fruiting body development. In addition, we summarize the research on a complex two-component system (Esp) that is important for the spatial regulation of sporulation.

The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1 Apart from the numerous studies that had focused on the biochemical characterization of BdhA, there was nothing known about the assimilation of (R)-3-HB in Pseudomonas, including the genetic regulation of bdhA expression. This study aimed to define the regulatory factors that govern or dictate the expression of the bdhA gene and (R)-3-HB assimilation in Pseudomonas aeruginosa PAO1. Importantly, expression of the bdhA gene was found to be specifically induced by (R)-3-HB in a manner dependent on the alternative sigma factor RpoN and the enhancer-binding protein PA2005.This mode of regulation was essential for the utilization of (R)-3-HB as a sole source of energy and carbon. However, non-induced levels of bdhA expression were sufficient for P. aeruginosa PAO1 to grow on (¡)-1,3-butanediol, which is catabolized through an (R)-3-HB intermediate. Because this is, we believe, the first report of an enhancer-binding protein that responds to (R)-3-HB, PA2005 was named HbcR for (R)-3-hydroxybutyrate catabolism regulator. Under starvation conditions, the PHA granule is degraded into its 3-hydroxy acid components, which can be used as sources of carbon and energy (Jendrossek & Handrick, 2002;Jendrossek et al., 1996).Pseudomonas species do not biosynthesize nor incorporate (R)-3-HB into their PHA reserves (Huisman et al., 1989; Timm & Steinbüchel, 1990). Nonetheless, these bacteria possess an NAD + -dependent dehydrogenase (BdhA) that converts (R)-3-HB into acetoacetate, thereby allowing these bacteria to use (R)-3-HB as a growth substrate (Feller et al., 2006;Ito et al., 2006;Mountassif et al., 2010). BdhA dehydrogenases have been biochemically characterized for some species of Pseudomonas, including P. putida (Feller et al., 2006;Paithankar et al., 2007)

Myxococcus xanthus has a large number of histidine kinase (HK) signal transduction proteins and many of these HKs are important for fruiting body development. Nla6S is an uncharacterized HK that lacks many of the conserved sequence motifs of typical HK proteins. In this study, we report that expression of the nla6S gene increases about sixfold during fruiting body development, that the Nla6S protein has the in vitro properties of HKs and that Nla6S is the prototype for a new family of HKs. To date, these Nla6-like HKs are found only in fruiting members of the Cystobacterineae suborder of the myxobacteria.

Dimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives in Pseudomonas aeruginosa PAO1. First, results from cell growth experiments showed that deletion of either sfnR2 or sfnG, a gene encoding a DMSO 2 -monooxygenase, significantly inhibits the ability of P. aeruginosa PAO1 to use DMSP, DMS, DMSO, and DMSO 2 as sulfur sources. Deletion of the sfnR1 or msuEDC genes, which encode a MSA desulfurization pathway, did not abolish the growth of P. aeruginosa PAO1 on any sulfur compound tested. Second, data collected from ␤-galactosidase assays revealed that the msuEDC-sfnR1 operon and the sfnG gene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression of sfnR2 is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of the msuEDC-sfnR1 operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression of msuEDC-sfnR1, sfnG, and other target genes involved in DMS-related metabolism in P. aeruginosa PAO1. IMPORTANCE Dimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, including Pseudomonas, Xanthomonas, and Azotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism in P. aeruginosa PAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.