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Proteus mirabilis is a dimorphic motile bacterium well known for its flagellum-dependent swarming motility over surfaces. In liquid, P. mirabilis cells are 1.5- to 2.0-μm swimmer cells with 4 to 6 flagella. When P. mirabilis encounters a solid surface, where flagellar rotation is limited, swimmer cells differentiate into elongated (10- to 80-μm), highly flagellated swarmer cells. In order for P. mirabilis to swarm, it first needs to detect a surface. The ubiquitous but functionally enigmatic flagellar basal body protein FliL is involved in P. mirabilis surface sensing. Previous studies have suggested that FliL is essential for swarming through its involvement in viscosity-dependent monitoring of flagellar rotation. In this study, we constructed and characterized Δ fliL mutants of P. mirabilis and Escherichia coli . Unexpectedly and unlike other fliL mutants, both P. mirabilis and E. coli Δ fliL cells swarm (Swr + ). Further analysis revealed that P. mirabilis Δ fliL cells also exhibit an alteration in their ability to sense a surface: e.g., Δ fliL P. mirabilis cells swarm precociously over surfaces with low viscosity that normally impede wild-type swarming. Precocious swarming is due to an increase in the number of elongated swarmer cells in the population. Loss of fliL also results in an inhibition of swarming at E. coli Δ fliL cells also exhibit temperature-sensitive swarming. These results suggest an involvement of FliL in the energetics and function of the flagellar motor.

Proteus mirabilis is a urinary tract pathogen and well known for its ability to move over agar surfaces by flagellum-dependent swarming motility. When P. mirabilis encounters a highly viscous environment, e.g., an agar surface, it differentiates from short rods with few flagella to elongated, highly flagellated cells that lack septa and contain multiple nucleoids. The bacteria detect a surface by monitoring the rotation of their flagellar motors. This process involves an enigmatic flagellar protein called FliL, the first gene in an operon ( fliLMNOPQR ) that encodes proteins of the flagellar rotor switch complex and flagellar export apparatus. We used a fliL knockout mutant to gain further insight into the function of FliL. Loss of FliL results in cells that cannot swarm (Swr − ) but do swim (Swm + ) and produces cells that look like wild-type swarmer cells, termed “pseudoswarmer cells,” that are elongated, contain multiple nucleoids, and lack septa. Unlike swarmer cells, pseudoswarmer cells are not hyperflagellated due to reduced expression of flaA (the gene encoding flagellin), despite an increased transcription of both flhD and fliA , two positive regulators of flagellar gene expression. We found that defects in fliL prevent viscosity-dependent sensing of a surface and viscosity-dependent induction of flaA transcription. Studies with fliL cells unexpectedly revealed that the fliL promoter, fliL coding region, and a portion of fliM DNA are needed to complement the Swr − phenotype. The data support a dual role for FliL as a critical link in sensing a surface and in the maintenance of flagellar rod integrity.

Silicibacter sp. strain TM1040, a member of the Roseobacter clade, forms a symbiosis with unicellular phytoplankton, which is inextricably linked to the biphasic "swim or stick" lifestyle of the bacteria. Mutations in flaC bias the population toward the motile phase. Renewed examination of the FlaC(-) strain (HG1016) uncovered that it is composed of two different cells: a pigmented type, PS01, and a nonpigmented cell, PS02, each of which has an identical mutation in flaC. While monocultures of PS01 and PS02 had few motile cells (0.6 and 6%, respectively), coculturing the two strains resulted in a 10-fold increase in the number of motile cells. Cell-free supernatants from coculture or wild-type cells were fully capable of restoring motility to PS01 and PS02, which was due to increased fliC3 (flagellin) transcription, FliC3 protein levels per cell, and flagella synthesis. The motility-inducing compound has an estimated mass of 226 Da, as determined by mass spectrometry, and is referred to as Roseobacter Motility Inducer (RMI). Mutations affecting genes involved in phenyl acetic acid synthesis significantly reduced RMI, while defects in tropodithietic acid (TDA) synthesis had marginal or no effect on RMI. RMI biosynthesis is induced by p-coumaric acid, a product of algal lignin degradation. When added to algal cultures, RMI caused loss of motility, cell enlargement, and vacuolization in the algal cells. RMI is a new member of the roseobacticide family of troponoid compounds whose activities affect roseobacters, by shifting their population toward motility, as well as their phytoplankton hosts, through an algicidal effect.

Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL , encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF , and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD 4 C 2 , and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD 4 C 2 , which ultimately controls swarmer cell differentiation.

The Escherichia coli flagellar master regulator, FlhD 4 C 2 , binds to the promoter regions of flagellar class II genes, yet, despite extensive analysis of the FlhD 4 C 2 -regulated promoter region, a detailed consensus sequence has not emerged. We used in vitro and in vivo experimental approaches to determine the nucleotides in the class II promoter, fliAp , required for the binding and function of FlhD 4 C 2 . FlhD 4 C 2 protects 48 bp (positions −76 to −29 relative to the σ 70 -dependent transcriptional start site) in the fliA promoter. We divided the 48-bp footprint region into 5 sections to determine the requirement of each DNA segment for the binding and function of FlhD 4 C 2 . Results from an in vitro binding competition assay between the wild-type FlhD 4 C 2 -protected fragment and DNA fragments possessing mutations in one section of the 48-bp protected region showed that only one-third of the 48 bp protected by FlhD 4 C 2 is required for FlhD 4 C 2 binding and fliA promoter activity. This in vitro binding result was also seen in vivo with fliA promoter- lacZ fusions carrying the same mutations. Only seven bases (A 12 , A 15 , T 34 , A 36 , T 37 , A 44 , and T 45 ) are absolutely required for the promoter activity. Moreover, A 12 , A 15 , T 34 , T 37 , and T 45 within the 7 bases are highly specific to fliA promoter activity, and those bases form an asymmetric recognition site for FlhD 4 C 2 . The implications of the asymmetry of the FlhD 4 C 2 binding site and its potential impact on FlhD 4 C 2 are discussed.

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria , and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles (∼50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.

The IX International Conference on Bacterial Locomotion and Signal Transduction (BLAST IX) was held from 14 to 19 January 2007 in Laughlin, NV, a town in the Mojave Desert on the Nevada-Arizona border near old Route 66 and along the banks of the Colorado River. This area is a home to rattlesnakes

The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda − ) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F , could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed.

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir -related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.

Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida , produced antibacterial compounds. Many non- Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter ) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non- Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.

Cold environments are common throughout the Galaxy. We are conducting a series of experiments designed to probe the low-temperature limits for growth in selected methanogenic and halophilic Archaea. This paper presents initial results for two mesophiles, a methanogen, Methanosarcina acetivorans, and a halophile, Halobacterium sp. NRC-1, and for two Antarctic cold-adapted Archaea, a methanogen, Methanococcoides burtonii, and a halophile, Halorubrum lacusprofundi. Neither mesophile is active at temperatures below 5 °C, but both cold-adapted microorganisms show significant growth at sub-zero temperatures (−2 °C and −1 °C, respectively), extending previous low-temperature limits for both species by 4–5 °C. At low temperatures, both H. lacusprofundi and M. burtonii form multicellular aggregates, which appear to be embedded in extracellular polymeric substances. This is the first detection of this phenomenon in Antarctic species of Archaea at cold temperatures. The low-temperature limits for both psychrophilic species fall within the temperature range experienced on present-day Mars and could permit survival and growth, particularly in sub-surface environments. We also discuss the results of our experiments in the context of known exoplanet systems, several of which include planets that intersect the Habitable Zone. In most cases, those planets follow orbits with significant eccentricity, leading to substantial temperature excursions. However, a handful of the known gas giant exoplanets could potentially harbour habitable terrestrial moons.

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.

The 54-kDa extracellular metalloprotease ZapA is an important virulence factor of uropathogenic Proteus mirabilis . While ZapA has the ability to degrade host immunoglobulins (Igs), the dramatic attenuation of virulence in ZapA mutants suggests that this enzyme may have a broader spectrum of activity. This hypothesis was tested by in vitro assays with purified ZapA and an array of purified protein or peptide substrates. The data reveal that many proteins found in the urinary tract are substrates of ZapA proteolysis, including complement (C1q and C3), cell matrix (collagen, fibronectin, and laminin), and cytoskeletal proteins (actin and tubulin). Proteolysis of IgA and IgG was significantly enhanced by conditions that denatured the Igs. It was discovered that the antimicrobial peptides human β-defensin 1 (hBD1) and LL-37 are readily cleaved by the enzyme. To the best of our knowledge, this is the first report of a bacterial protease capable of cleaving hBD1, a component of the human renal tubule innate immune response. Proteolysis of hBD1 resulted in ca. six peptides, while proteolysis of LL-37 resulted in at least nine products. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the molecular masses of the reaction products indicated that ZapA preferred no distinct peptide bond. The antimicrobial activity of hBD1 and LL-37 was significantly reduced following ZapA treatment, suggesting that proteolysis results in inactivation of these peptides. The data suggest that a function of ZapA during urinary tract infections is the proteolysis of antimicrobial peptides associated with the innate immune response.

The α-proteobacteria phylogenetically related to the Roseobacter clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP). Silicibacter sp. strain TM1040, isolated from a DMSP-producing Pfiesteria piscicida dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate. TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with P. piscicida through a chemotactic response to compounds produced by its dinoflagellate host. A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040. These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80°C. To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants. The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates. Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates. The implication of Silicibacter sp. strain TM1040 chemotaxis in establishing and maintaining its interaction with P. piscicida is discussed.

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. The VPI can excise from the chromosome and form extrachromosomal circular excision products. The VPI is 41.2-kb in size and encodes 29 potential proteins, several of which have no known function and whose regulation is not well understood. To determine the transcriptional organization of the tagA-orf2-orf3-mop-tagD region located at the 5'-(left) end of the VPI, we used reverse-transcriptase-PCR (RT-PCR), Northern blot analysis and DNA sequencing. RT-PCR primers were designed to transcribe and amplify regions spanning two or more open reading frames so as to establish the transcriptional organization. RT-PCR and Northern blot results demonstrated that the tagA-tagD region is transcribed as a polycistronic message and organized into several potential operons including tagA-orf2, orf3-mop, orf3-mop-tagD and tagD alone. Transcriptional lacZ fusions supported the existence of a promoter upstream of orf3 that was toxT-dependent. Interestingly, our data suggests that the orf3 promoter can drive the expression of either a long transcript (orf3-mop-tagD) or a short transcript (orf3-mop) without tagD. Our data also suggests that tagD can be expressed from two different promoters and that tagD is either transcribed alone or co-expressed with orf3-mop under certain conditions. These studies provide new insight into the genetic structure, transcriptional organization and regulation of a cluster of virulence genes on the VPI of epidemic V. cholerae.

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria -like ( Cryptoperidiniopsis ) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 μM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 μM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.

DNA microarrays and two-dimensional (2-D) gel electrophoresis were utilized to analyze the global effect of bile on transcription and protein synthesis in Salmonella enterica serovar Typhimurium. Two bile-regulated proteins, YciF and PagC, were identified by 2-D gel electrophoresis and mass spectrometry fingerprinting. The operon yciGFE-katN demonstrated increased transcriptional activity in the presence of bile. While this operon has previously been shown to be RpoS-regulated, data from this study suggested that yciGFE-katN is regulated by bile independent of RpoS. The PhoP-PhoQ-regulated PagC is decreased in the presence of bile. Characterization of the untranslated leader of pagC demonstrated that a 97-bp region is necessary for the bile-mediated repression of this promoter. Analysis of data from the DNA microarray revealed an effect of bile on important global mechanistic pathways in S. enterica serovar Typhimurium. Genes involved in type III secretion-mediated invasion of epithelial cells demonstrated an overall repression of transcription in the presence of bile, corroborating previously reported data from this laboratory [Infect. Immun. 68 (2000) 6763]. In addition, bile-mediated transcriptional repression of genes involved in flagellar biosynthesis and motility was observed. These data further demonstrate that bile is an important environmental signal sensed by Salmonella spp. and that bile plays a role in regulating bacterial gene expression in multiple virulence-associated pathways.

Pfiesteria piscicida and Pfiesteria shumwayae are estuarine dinoflagellates thought to be responsible for massive fish deaths and associated human illnesses in the southeastern United States. These dinoflagellates are described as having a complex life cycle involving flagellated zoospores, cysts, and amoeboid stages. Although no Pfiesteria toxin has been identified, certain strains of these dinoflagellates are thought to produce a water-soluble toxin that can kill fish and cause human illness. Recent reports show no evidence for amoeboid stages and indicate that a much more simplified life cycle exists. In addition, researchers have shown that P. shumwayae only kills fish through direct contact that does not necessarily involve the production of one or more toxins. This review summarizes these and other recent findings with an emphasis on establishing basic facts regarding the toxicity and life history of Pfiesteria dinoflagellates.

Quorum sensing regulates the expression of virulence factors in a wide variety of pathogenic bacteria. This study has shown that Proteus mirabilis harbours a homologue of luxS, a gene required for the synthesis of the quorum sensing autoinducer 2 (AI-2). AI-2 activity is expressed during and is correlated with the initiation of swarming migration on agar surfaces. The P. mirabilis luxS locus was cloned and a LuxS− strain constructed by allelic-exchange mutagenesis. While lacking AI-2 activity, a null mutation in luxS, however, did not affect swimming or swarming motility, swarmer cell differentiation, or virulence in a mouse model of ascending urinary tract infection.

The description of the heterotrophic dinoflagellate Pfiesteriapiscicida as the causative agent of fish lesions and deaths alongthemid-Atlantic estuaries has revealed the need for bioassays to assess itspotential toxigenicity. We designed a bioassay in which fish are exposed toclonal dinoflagellate strains or environmental consortia (e.g. environmentalwater or sediment samples) in 750-mL culture flasks, and examinedthe relationships among dinoflagellate proliferation profiles, the presence ofP. piscicida and fish deaths. Assay development andcharacterization were accomplished with dinoflagellate clonal cultures(P. piscicida, Karlodinium micrum,Prorocentrum minimum, CCMP1828, CCMP1829, and CCMP1834)co-incubated with sets of two young adult sheepshead minnows(Cyprinodon variegatus). Variables characterized includedwater quality (pH, dissolved oxygen, ammonia, nitrate and nitriteconcentrations), the effect of the presence of fish on the proliferation andcompositions of protist (dinoflagellate, protozoa, diatom) and bacterialpopulations, and time of fish death. The presence of fish in experimentalflasksinduced proliferation of P. piscicida andCryptoperidiniopsis sp. (but not K.micrum or P. minimum) with populationsrising between days 6 and 10, and declining 4 to 5 days later. Some fish deathsoccurred when or soon after P. piscicida cell numberswere maximal. We conclude that this assay enables the assessment of acuteeffects of ichthyocidal dinoflagellates on fish during the first 10 days (StageA) of the experimental course. Fish deaths during the subsequent 10 to 20 days(Stage B) may be attributed to the proliferation of ichthyocidaldinoflagellates, pathogenic bacteria and/or deteriorating water quality,whereasthose beyond a period of approximately three weeks (Stage C) can be mostcertainly attributed to deteriorated water quality. Application of the flaskassay to environmental samples [n=53] yielded fish deaths in all three stages:A, 78%; B, 11%, and C, 2%, with fish still living in 9% of the sample waterstested at the conclusion of the experiment beyond three weeks. The majority ofsamples that resulted in fish death in stage A tested positive for P.piscicida by PCR. If implemented with cautious interpretation, thisassay should prove useful in monitoring blooms for the presence of P.piscicida and other dinoflagellate s pecies potentially harmful tofish.

Formation of a bacterial biofilm is a developmental process that begins when a cell attaches to a surface, but how does a bacterial cell know it is on or near a surface in the first place? The phase of this 'swim-or-stick' switch is determined by a sensory transduction mechanism referred to as surface sensing, which involves the rotating bacterial flagellum. This review explores six bacterial species as models of flagellar mechanosensing of surfaces to understand the current state of our knowledge and the challenges that lie ahead. A common link between these bacteria is a requirement for the proper function of the flagellar motor stators that channel ions into the cell to drive flagellar rotation. Conditions that affect ion flow act as a signal that, ultimately, controls the master transcriptional regulatory circuits controlling the flagellar hierarchy and biofilm formation.

The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.

Summary Bacteria live either as independent planktonic cells or as members of surface-attached communities called biofilms. Motility and biofilm development are mutually exclusive events, and control of the phase of this ‘swim-or-stick’ switch involves the ability of the bacterium to sense and respond appropriately to a surface. Cairns et al. (2013) report that the Bacillus subtilis flagellum functions in surface-sensing. Using mutants of B. subtilis that prevent flagellum rotation, they measured the expression and activity of DegU, the response regulator of the two-component DegS–DegU circuit. DegU activity and degU transcription increased when flagellum rotation was prevented, and were dependent on the DegS kinase. Inhibiting flagellar rotation by overexpressing the EpsE flagellar ‘clutch’ or addition of anti-flagellin antiserum also increased degU transcription and activity. These results suggest B. subtilis senses restriction of flagellum rotation as the cell nears a surface. Inhibition of the flagellum activates the DegS–DegU circuit to turn on biofilm formation, i.e. the flagellum is acting as a mechanosensor of surfaces. B. subtilis joins an ever-expanding group of bacteria, including species of Vibrio, Proteus and Caulobacter that use the flagellum as a surface sensor.

Proteus mirabilis is best known for its pattern of swarming differentiation on agar plates, as well as for its association with the development of renal stones in patients with urinary tract infection. Urease and flagella appear to contribute most significantly to virulence, with fimbriae playing a more subtle role, whereas hemolysin does not appear to contribute significantly to pathogenesis.

Swarmer cell differentiation is a complex process involving the activity of many gene products. In this report, we characterized the genetic locus of Tn5 insertion in each of 12 mutants defective in swarmer cell elongation. The mutations fell into four categories affecting either flagellar biosynthesis or energetics, lipopolysaccharide and cell wall biosynthesis, cellular division, or proteolysis of peptides.

Fish mycobacteriosis is a chronic bacterial disease that has the potential for infecting most fish species from both freshwater and saltwater habitats. The causative agents of fish mycobacteriosis are three species of Mycobacterium: M. chelonae, M. fortuitum, and M. marinum. Infections of fish by these bacteria result in a slow, degenerative disease with symptoms that include wasting, fading of color, exophthalmia, and frequently, granulomas and other types of skin ulcers. The disease is transmissible from fish to fish and also from fish to man, where the bacteria produce a potentially serious medical condition. Little is known about the molecular biological aspects of the disease or the host-pathogen interactions. Using the knowledge gained from recent advances in the study of M. tuberculosis and other human mycobacterial pathogens, we suggest a set of strategies to begin a molecular biological study of fish mycobacteriosis. Although no conclusions may be drawn at this early stage in understanding this disease, the prospects are very good that the techniques of molecular biology will shed light on this longstanding and misunderstood disease.

The overproduction of flagella is a distinguishing characteristic of Proteus mirabilis swarmer cell differentiation. The synthesis of flagellin, the principal protein composing the flagellar filament, is coordinately regulated as part of a larger regulon of genes whose expression is a prerequisite in urinary pathogenesis. In this report, the regulation of expression of the flaA locus, comprising flaA and flaB, two tandemly linked and nearly identical copies of flagellin-encoding genes, is examined. Transcriptional expression studies reveal that flaA, but not flaB, is expressed by wild-type cells, and flaA transcription increases eightfold during differentiation. The flaA transcriptional start site for both swimmer and swarmer cells was determined to be located at a guanine, 8 bases downstream of the flaA sigma 28 promoter. FlaA- mutants are nonmotile and undifferentiated and do not synthesize flagellin, while FlaB- mutants are wild type, thus verifying that FlaA is the sole flagellin produced by wild-type cells and that flaB is silent. FlaA- mutants frequently revert to a Mot+ phenotype that is antigenically distinct from that of wild-type cells. Southern blot analysis of the flaA Mot+ revertants reveals a deletion of between 2 and 7kb in the flaA locus. Biochemical analyses of revertant flagellin indicate major changes in protein size and composition but conservation of the first 28 N-terminal residues. The result of this process is to produce an antigenically distinct flagellum that may be significant in ensuring the survival of P. mirabilis during pathogenesis.

Surface-induced overproduction of flagellin is one of the hallmarks of Proteus mirabilis swarmer cell differentiation. In this study, we analyzed the nucleotide (nt) and amino acid (aa) sequences, and expression of the P. mirabilis flagellin-encoding gene (fliC) region. The nt sequence analysis of a 3567-bp region reveals three ORFs, each with homology to known Escherichia coli flagellar genes. The first ORF corresponds to fliD, the gene encoding the flagellar filament capping protein, FliD (HAP2). The second and third ORFs are highly homologous to each other and to fliC genes from many other Gram- bacteria. To distinguish between the two alleles, we have designated these genes fliC1 and fliC2. Sequences highly homologous to promoter sites for the alternate sigma factor of RNA polymerase, sigma 28, are found 5' to the start of each gene. Additionally, both fliC1 and fliC2 have a conserved direct tandem repeat (DTR) sequence upstream from the sigma 28 promoter that may have functional significance in the transcriptional control of fliC expression during swarmer cell differentiation. Both FliC1 and FliC2 were produced in E. coli, but only FliC1 could complement FliC- mutants of E. coli. Southern hybridization data indicate the presence of fliC1 and fliC2 in six distinct P. mirabilis strains, indicating that multiple flagellin-encoding genes are common in P. mirabilis. Hybridization data also suggest the presence of a third flagellin-encoding gene, fliC3, in all isolates. The possible significance of multiple fliC in swarmer cell differentiation is discussed.

Silicibacter sp. TM1040, a member of the marine Roseobacter clade, produces the antibiotic and quorum signaling molecule tropodithietic acid (TDA), encoded by tdaABCDEF . Here, we showed that an LysR-type transcriptional regulator, TdaA, is a positive regulator of tdaCDE gene expression and binds to the tdaC promoter region.

The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tda ABCDEF ( tdaA-F ), as well as six additional genes ( cysI , malY , paaIJK , and tdaH ). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaC p :: lacZ transcriptional fusion was placed in 11 of the 12 Tda − mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda − mutants— tdaA and tdaH failed to respond—by placing wild-type (Tda + ) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

Members of the Roseobacter clade of alpha-proteobacteria are among the most abundant and ecologically relevant marine bacteria. Bacterial isolates and gene sequences derived from this taxonomic lineage have been retrieved from marine environments ranging from sea ice to open ocean mixed layer to tropical coral reefs, and in ecological niches ranging from free-living plankton to sponge symbiont to biofilm pioneer. Although roseobacters are cosmopolitan in the marine environment, their numbers and activity significantly rise with increases in the population density of phytoplankton [1,2], suggesting that these bacteria are highly adapted to engage in these symbioses. This review examines the molecules and phenotypes of roseobacters that are important in establishing and maintaining the symbioses between roseobacters and phytoplankton.

Silicibacter sp. TM1040 is a member of the marine Roseobacter clade of Alphaproteobacteria that forms symbioses with unicellular eukaryotic phytoplankton, such as dinoflagellates. The symbiosis is complex and involves a series of steps that physiologically change highly motile bacteria into cells that readily form biofilms on the surface of the host. The initial phases of symbiosis require bacterial motility and chemotaxis that drive the swimming bacteria toward their planktonic host. Cells lacking wild-type motility fail to establish biofilms on host cells and do not produce effective symbioses, emphasizing the importance of understanding the molecular mechanisms controlling flagellar biosynthesis and the biphasic “swim-or-stick” switch. In the present study, we used a combination of bioinformatic and genetic approaches to identify the genes critical for swimming of Silicibacter sp. TM1040. More than 40 open reading frames with homology to known flagellar structural and regulatory genes were identified, most of which are organized into approximately eight operons comprising a 35.4-kb locus, with surprising similarity to the fla2 locus of Rhodobacter sphaeroides . The genome has homologs of CckA, CtrA, FlbT, and FlaF, proteins that in Caulobacter crescentus regulate flagellum biosynthesis. In addition, we uncovered three novel genes, flaB , flaC , and flaD , which encode flagellar regulatory proteins whose functions are likely to involve regulation of motor function (FlaD) and modulation of the swim-or-stick switch (FlaC). The data support the conclusion that Silicibacter sp. TM1040 uses components found in other Alphaproteobacteria , as well as novel molecular mechanisms, to regulate the expression of the genes required for motility and biofilm formation. These unique molecular mechanisms may enhance the symbiosis and survival of Roseobacter clade bacteria in the marine environment.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.

Our knowledge of pathogenesis has benefited from a better understanding of the roles of specific virulence factors in disease. To determine the role of the virulence factor ZapA, a 54-kDa metalloproteinase of Proteus mirabilis , in prostatitis, rats were infected with either wild-type (WT) P. mirabilis or its isogenic ZapA − mutant KW360. The WT produced both acute and chronic prostatitis showing the typical histological progressions that are the hallmarks of these diseases. Infection with the ZapA − mutant, however, resulted in reduced levels of acute prostatitis, as determined from lower levels of tissue damage, bacterial colonization, and inflammation. Further, the ZapA − mutant failed to establish a chronic infection, in that bacteria were cleared from the prostate, inflammation was resolved, and tissue was seen to be healing. Clearance from the prostate was not the result of a reduced capacity of the ZapA − mutant to form biofilms in vitro. These finding clearly define ZapA as an important virulence factor in both acute and chronic bacterial prostatitis.

The shallow depth of field of conventional microscopy hampers analyses of 3D swimming behavior of fast dinoflagellates, whose motility influences macroassemblages of these cells into often-observed dense “blooms.” The present analysis of cinematic digital holographic microscopy data enables simultaneous tracking and characterization of swimming of thousands of cells within dense suspensions. We focus on Karlodinium veneficum and Pfiesteria piscicida , mixotrophic and heterotrophic dinoflagellates, respectively, and their preys. Nearest-neighbor distance analysis shows that predator and prey cells are randomly distributed relative to themselves, but, in mixed culture, each predator clusters around its respective prey. Both dinoflagellate species exhibit complex highly variable swimming behavior as characterized by radius and pitch of helical swimming trajectories and by translational and angular velocity. K. veneficum moves in both left- and right-hand helices, whereas P. piscicida swims only in right-hand helices. When presented with its prey ( Storeatula major ), the slower K. veneficum reduces its velocity, radius, and pitch but increases its angular velocity, changes that reduce its hydrodynamic signature while still scanning its environment as “a spinning antenna.” Conversely, the faster P. piscicida increases its speed, radius, and angular velocity but slightly reduces its pitch when exposed to prey ( Rhodomonas sp.), suggesting the preferred predation tactics of an “active hunter.”

Summary Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot–) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla–), while CtrA– cells possess flagella, but have reduced motility due to increased cell length. All three Mot– mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla– mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040–P. piscicida interaction.

Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL , fliF , and fliG , encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL − mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL + in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL + was expressed in Escherichia coli . These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB . These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.

Proteus mirabilis, a Gram-negative urinary tract pathogen, has two highly homologous, tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a σ 28 promoter, while flaB is a silent allele. Previous studies have demonstrated the presence of a family of hybrid flagellin genes, referred to as flaAB. These genes are composed of the 5′ end of flaA and the 3′ end of flaB, and are produced through excision of the intervening DNA between the two genes. Although the existence of flaAB DNA has been documented, it was not known if transcription of flaAB occurs in wild-type P. mirabilis. In this study, proof of flaAB transcription was obtained from a combination of RNA dot-blots and RT-PCR assays using specific primers and probes for flaAB and flaA. The RNA data were further supported by the demonstration of phenotypic switching of the locus using a FlaAB-detector strain. The results show that flaAB mRNA is transcribed and is 1/64 as abundant as flaA in the population of wild-type cells, suggesting that flaAB constitutes 1·0–1·5 % of the total flagellin message. Nucleotide sequence analysis of flaAB products produced by RT-PCR from the wild-type confirms previous reports of a variable fusion site between flaA and flaB resulting in a hybrid flagellin transcript. These data support the hypothesis that the production of FlaAB is integral to the physiology of P. mirabilis.

Proteus mirabilishas two tandemly arranged flagellin-encoding genes,flaAandflaB.flaAis transcribed from aσ28promoter, whileflaBis silent.flaAandflaBcan undergo reversible rearrangement to produce a set of hybrid genes referred to asflaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism forP. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-typeP. mirabiliswas compared with that of a strain, DF1003, locked into the FlaAB+hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF1003 is also significantly faster than the wild-type at pH 5·2, 5·8 and 8·2, and at 5 and 10 % polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5·8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage toP. mirabilisin conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.

Quorum sensing regulates the expression of virulence factors in a wide variety of pathogenic bacteria. This study has shown that Proteus mirabilis harbours a homologue of luxS, a gene required for the synthesis of the quorum sensing autoinducer 2 (AI-2). AI-2 activity is expressed during and is correlated with the initiation of swarming migration on agar surfaces. The P. mirabilis luxS locus was cloned and a LuxS(-) strain constructed by allelic-exchange mutagenesis. While lacking AI-2 activity, a null mutation in luxS, however, did not affect swimming or swarming motility, swarmer cell differentiation, or virulence in a mouse model of ascending urinary tract infection.

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four 'Pfiesteria-like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the alpha-3 or SAR83 cluster.

Publisher Summary This chapter discusses that transposon mutagenesis is a powerful tool to generate mutants that are defective in surface sensing, swarmer cell differentiation, and biofilm development. When coupled with transcriptional fusion to a promoterless reporter gene, transposon insertion into target genes can provide valuable information about the signals required for surface-induced gene expression. One possible limitation in using a transposon-reporter gene construct for gene expression studies is the large size of the reporter component, lac genes in mini-Mu lac and lux genes in mini-Mu lux add substantially to the size of the transposon. If the functional loss of the target gene is deleterious to the organism, then mutations in this gene will not be detected. This is a limitation of any study in which random mutagenesis is used to identify important genetic elements. A different reporter system is a variant of green fluorescent protein (GFPuv) from Aequorea victoria . Fluorescence production by GFP is species independent and does not require any substrate, cofactor, or other auxiliary genes. It is possible to construct an N- or C-terminal fusion protein with the target gene, while retaining the reporter activity of the GFP and function of the target gene. Bacterial colonies expressing GFPuv can be detected on agar, using a UV transilluminator. This system thus has the advantage of the transposon-generated transcriptional fusions, yet retains the activity of the target gene, and lacks polar effects.