Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway

Density-dependent expression of luminescence in Vibrio harveyl is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably β-hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyl.     

Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) In the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 Insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO∼ mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.     

Sequence and Function of LuxU: a Two-Component Phosphorelay Protein That Regulates Quorum Sensing in Vibrio harveyi

Vibrio harveyi regulates the expression of bioluminescence (lux) in response to cell density, a phenomenon known as quorum sensing. In V. harveyi, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.     

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

Aims:  To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Methods and Results:  Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. Conclusions:  There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. Significance and Impact of the Study:  The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.     

Synthesis of the lux gene autoinducer in Vibrio fischeri is positively autoregulated

The enzymes for luminescence in Vibrio fischeri are induced only after the accumulation of a sufficient concentration of a metabolic product (the autoinducer) generated by the bacteria themselves. Genetic analyses by others have previously suggested that biosynthesis of the autoinducer is catalyzed by a single gene product (autoinducer synthetase) presumably from precursors typically present in the bacterial cell. Also, the biosynthesis was predicted to be autocatalytic such that in the presence of autoinducer, more autoinducer synthetase should be produced. We have directly tested these predictions and found that autoinducer synthesis is indeed positively autoregulated. In addition, we have demonstrated autoinducer synthesis in vitro and have tentatively identified the substrates of autoinducer synthetase as S-adenosylmethionine and 3-oxohexanoyl coenzyme A.Abbreviations AdoMet S-adenosylmethionine - AI autoinducer, i.e. 3-oxohexsanoyl homoserine lactone - C-10 decanoyl homoserine lactone - HPLC high performance liquid chromatography - LM luminescence medium - LM-BT luminescence medium without tryptone - LU light units - 3-oxo 3-oxohexanoyl-coenzyme A - SWC sea water complete medium     

Quorum sensing in Vibrio fischeri: evidence that S-adenosylmethionine is the amino acid substrate for autoinducer synthesis.

Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri autoinducer.     

Neural crest cell‐specific inactivation of Nipbl or Mau2 during mouse development results in a late onset of craniofacial defects

Nipbl (Scc2) and Mau2 (Scc4) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell‐specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl;Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner. genesis 52:687–694, 2014. © 2014 Wiley Periodicals, Inc.     

Analogs of the autoinducer of bioluminescence inVibrio fischer

The enzymes for luminescence inVibrio fischeri are induced only when a sufficient concentration of a metabolic product (autoinducer) specifically produced by this species accumulates. It has previously been shown that the autoinducer is 3-oxohexanoyl homoserine lactone and that it enters the cells by simple diffusion. To further study the mechanism of induction, we have synthesized several analogs of the autoinducer. The analogs were tested withV. fischeri for their inducing activity and for their ability to inhibit the action of the natural autoinducer. The compounds were found to display various combinations of inducing and inhibiting abilities. None of the compounds tested appeared to have any effect on cells ofV. harveyi strain MAV orPhotobacterium leiognathi strain 721, but several of the compounds decreased light output byP. phosphoreum strain 8265. These studies show that 1) the site of action of the autoinducer is not highly sterically constrained 2) the autoinducers of other species of luminous bacteria are likely to be quite different from that ofV. fischeri and 3) a simple mode in which one autoinducer molecule binds to a single receptor protein site and thus initiates luciferase synthesis is inadequate. The analogs should prove useful in the study of the binding site and mode of action of the autoinducer.Abbreviations SWC sea water complete     

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum‐sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI‐independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester‐containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4‐mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild‐type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.     

Determinants governing ligand specificity of the Vibrio harveyi LuxN quorum‐sensing receptor

Quorum sensing is a process of bacterial cell–cell communication that relies on the production, release and receptor‐driven detection of extracellular signal molecules called autoinducers. The quorum‐sensing bacterium Vibrio harveyi exclusively detects the autoinducer N‐((R)‐3‐hydroxybutanoyl)‐L‐homoserine lactone (3OH‐C4 HSL) via the two‐component receptor LuxN. To discover the principles underlying the exquisite selectivity LuxN has for its ligand, we identified LuxN mutants with altered specificity. LuxN uses three mechanisms to verify that the bound molecule is the correct ligand: in the context of the overall ligand‐binding site, His210 validates the C₃ modification, Leu166 surveys the chain‐length and a strong steady‐state kinase bias imposes an energetic hurdle for inappropriate ligands to elicit signal transduction. Affinities for the LuxN kinase on and kinase off states underpin whether a ligand will act as an antagonist or an agonist. Mutations that bias LuxN to the agonized, kinase off, state are clustered in a region adjacent to the ligand‐binding site, suggesting that this region acts as the switch that triggers signal transduction. Together, our analyses illuminate how a histidine sensor kinase differentiates between ligands and exploits those differences to regulate its signaling activity.     

The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages

Legionella pneumophila, the causative agent of Legionnaires’ disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular lifestyle is the ability of the organism to replicate within a specialized phagosome which does not fuse with Iysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome–Iysosome fusion. In a previous study, a 12kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-Iysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-Iysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1–2.4kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have Inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.