Loss of FliL alters Proteus mirabilis surface sensing and temperature-dependent swarming.

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 ΔfliL mutants of P. mirabilis and Escherichia coli. Unexpectedly and unlike other fliL mutants, both P. mirabilis and E. coli ΔfliL cells swarm (Swr(+)). Further analysis revealed that P. mirabilis ΔfliL cells also exhibit an alteration in their ability to sense a surface: e.g., ΔfliL 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 <30°C. E. coli ΔfliL cells also exhibit temperature-sensitive swarming. These results suggest an involvement of FliL in the energetics and function of the flagellar motor.

When the swimming gets tough, the tough form a biofilm.

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.

A novel inducer of Roseobacter motility is also a disruptor of algal symbiosis.

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.

Activity of Proteus mirabilis FliL is viscosity dependent and requires extragenic DNA.

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.

Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation.

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.

TdaA regulates Tropodithietic acid synthesis by binding to the tdaC promoter region.

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.

Refining the binding of the Escherichia coli flagellar master regulator, FlhD4C2, on a base-specific level.

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.

Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

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 tdaABCDEF (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 tdaCp::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.

Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment.

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.

Genetic determinants of Silicibacter sp. TM1040 motility.

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.

ZapA, a virulence factor in a rat model of Proteus mirabilis-induced acute and chronic prostatitis.

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.

Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters.

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.

Occurrence and expression of gene transfer agent genes in marine bacterioplankton.

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 ( approximately 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.

Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene.

An agarase gene (agrA) was isolated by cloning genomic DNA prepared from Pseudomonas atlantica. The agarase activity in recombinant Escherichia coli was found in cell-free culture supernatants and could pass through a 0.45-mum-pore-size membrane separating cells from agar, suggesting that the gene product was exported in E. coli. The enzyme was specific for agar and agarose and did not digest alginate or carrageenan. Mutations generated by transposon mini-Mu d1(lacZ Km) were used to define the agrA coding region, as well as the direction of transcription of the gene. A procedure was developed to produce a P. atlantica agrA mutant. This required construction of an agrA::kan insertion mutation in vitro and subsequent introduction of the defect into the chromosome of P. atlantica by recombinational exchange. Transformation of P. atlantica with plasmids containing agrA::kan utilized a Tris-polyethylene glycol 6000-CaCl(2) treatment for making competent cells. Replacement of wild-type agrA with agrA::kan resulted in loss of agarase activity. Uses of the agrA gene probe and an Agr mutant for environmental studies are discussed.

Pfiesteria piscicida, P. shumwayae, and other Pfiesteria-like dinoflagellates.

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.

Transcriptional analysis and operon structure of the tagA-orf2-orf3-mop-tagD region on the Vibrio pathogenicity island in epidemic V. cholerae.

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.

Biofilms, flagella, and mechanosensing of surfaces by bacteria.

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.

Molecular mechanisms underlying roseobacter-phytoplankton symbioses.

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.

Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions.

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.