SummarySerratia marcescens SS-1 produces at least four N -acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homoserine lactone (3-oxo-C6-HSL), N -hexanoyl-(C6-HSL), N -heptanoyl (C7-HSL) and N -octanoyl-(C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactantmediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT . spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.

Serratia marcescens swarms at 30°C but not at 37°C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37°C but also earlier and faster than the parent strain swarms at 30°C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertiondeletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37°C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30°C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30°C or 37°C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.

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Swarming motility is a multicellular phenomenon comprising population migration across surfaces by specially differentiated cells. In Serratia marcescens, a network exists in which the flhDC flagellar regulatory master operon, temperature, nutrient status, and quorum sensing all contribute to the regulation of swarming motility. In this study, the rsmA (repressor of secondary metabolites) gene (hereafter rsmA_Sm) was cloned from S. marcescens. The presence of multicopy, plasmid-encoded rsmA_Sm expressed from its native promoter in S. marcescens inhibits swarming. Synthesis of N-acylhomoserine lactones, presumably by the product of smaI (a luxI homolog isolated from S. marcescens), was also inhibited. Knockout of rsmA_Sm on the S. marcescens chromosome shortens the time before swarming motility begins after inoculation to an agar surface. A single copy of the chromosomal PrsmA_Sm::luxAB reporter of rsmA_Smtranscription was constructed. Using this reporter, the roles of the flhDC flagellar regulatory master operon, temperature and autoregulation in the control of rsmA_Sm expression were determined. Our findings indicate that RsmA_Sm is a component of the complex regulatory network that controls swarming.

We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluations were investigation of cell division, flagellar synthesis and regulation of the expression of nuclease (encoded by the nucA_Sm gene, one of the virulence factors). Interruption of the chromosomal flhDC operon in S. marcescens CH-1 resulted in aberrant cell division and loss of nuclease and flagella. Expression of nucA_Sm and other mutated phenotypes was restored in the flhDC mutant by the induction of overexpression of flhDC in a multicopy plasmid. Multicopied flhDC also induced the formation of differentiated cells (polyploid aseptate cells with oversynthesis of peritrichous flagella) in broth culture using minimal growth medium. Expression of the flhDC operon showed positive autoregulation, and was growth phase dependent (upregulated in early log phase). In addition, flhDC expression was inhibited when the temperature increased from 30 to 37°C, and when osmolarity was increased, but was not influenced by glucose catabolite repression. These results show that FlhD/FlhC is a multifunctional transcriptional activator involved in the process of cell differentiation, swarming and virulence factor expression.

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin Sm ) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin Sm gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin Sm gene in S. marcescens CH-1. The S. marcescens CH-1 pirin Sm gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin Sm mutant. Concomitantly, the cellular NADH/NAD ؉ ratio increased in the pirin Sm mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin Sm gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin Sm is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.The protein pirin is widely found in mammals, plants, fungi, and also prokaryotic organisms (32). While the cellular functions of pirin show diversity and pirin homologs play important roles in a number of different biological processes, cellular localization of pirin is not restricted to specific compartments. In eukaryotes, pirin was initially isolated through a yeast twohybrid screen from the HeLa cell cDNA library and is localized within cell nuclei; it acts as an interactor with nuclear factor

Aim:  To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli. Method and Results:  The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose PBAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring PBAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions:  The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study:  We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes.

Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDC Sm and hag Sm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a twocomponent system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDC Sm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDC Sm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssBϳP) binding site by an electrophoretic mobility shift assay showed direct interaction of RssBϳP, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssBϳP binding site is located between base pair positions ؊341 and ؊364 from the translation start codon ATG in the flhDC Sm promoter region. The binding site overlaps with the P2 "؊35" promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssBϳP binds to the flhDC Sm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDC Sm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.Swarming is a bacterial population surface translocation behavior demonstrated in a wide range of diverse bacterial genera and species (2,12,14). In Serratia spp., swarming requires close interactions between the environment and the bacterial cells, as well as among the cells, in order to develop a high degree of complex cell coordination within the swarming colony (2,9,13,26,33,35). Previous studies on the regulation of swarming showed that bacterial flagellar, quorum-sensing, and two-component systems are important for swarming (3,7,33). Among these, flagellar motility, which is one of the essential factors for bacterial swarming, is controlled by the flagellar system, comprising large and complex regulons (4, 9). Studies with the flagellar systems of Escherichia coli and Salmonella enterica serovar Typhimurium have identified around 50 genes organized into three hierarchical transcriptional classes. At the top of the hierarchical cascade is the class I master operon flhDC (4). The FlhD 2 C 2 complex is a transcriptional activator of 70 -dependent transcription from class II promoters (4). Thus, activation of the whole set of flagellar motility genes depends mainly on the expression of flhDC.Serratia marcescens cells swarm at 30°C but not at 37°C (15).In a previous study utilizing a mini-Tn5 mutagenesis approach, we had ...