Valerie Stout

Role of RcsF in Signaling to the Rcs Phosphorelay Pathway in Escherichia coli

The rcs phosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of the yojN gene, now renamed rcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both the rprA and cps promoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting this signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of an rcsC allele, rcsC137, that leads to high-level constitutive expression of both cps and rprA suggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.

Molecular Genetics and Genomic Analysis of Scytonemin Biosynthesis in Nostoc punctiforme ATCC 29133

The indole-alkaloid scytonemin is the most common and widespread sunscreen among cyanobacteria. Previous research has focused on its nature, distribution, ecology, physiology, and biochemistry, but its molecular genetics have not been explored. In this study, a scytonemin-deficient mutant of the cyanobacterium Nostoc punctiforme ATCC 29133 was obtained by random transposon insertion into open reading frame NpR1273. The absence of scytonemin under conditions of induction by UV irradiation was the single phenotypic difference detected in a comparative analysis of the wild type and the mutant. A cause-effect relationship between the phenotype and the mutation in NpR1273 was demonstrated by constructing a second scytoneminless mutant through directed mutagenesis of that gene. The genomic region flanking the mutation revealed an 18-gene cluster (NpR1276 to NpR1259). Four putative genes in the cluster, NpR1274 to NpR1271, with no previously known functions, are likely to be involved in the assembly of scytonemin. Also in this cluster, there is a redundant set of genes coding for shikimic acid and aromatic amino acid biosynthesis enzymes, leading to the production of tryptophan and tyrosine, which are likely to be biosynthetic precursors of the sunscreen.

Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli.

ABSTRACT Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.

Gene Expression Patterns Associated with the Biosynthesis of the Sunscreen Scytonemin in Nostoc punctiforme ATCC 29133 in Response to UVA Radiation

Under exposure to UV radiation, some cyanobacteria synthesize sunscreen compounds. Scytonemin is a heterocyclic indole-alkaloid sunscreen, the synthesis of which is induced upon exposure to UVA (long-wavelength UV) radiation. We previously identified and characterized an 18-gene cluster associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133; we now report on the expression response of these genes to a step-up shift in UVA exposure. Using quantitative PCR on cDNAs from the N. punctiforme transcriptome and primers targeting each of the 18 genes in the cluster, we followed their differential expression in parallel subcultures incubated with and without UVA. All 18 genes are induced by UVA irradiation, with relative transcription levels that generally peak after 48 h of continuous UVA exposure. A five-gene cluster implicated in the process of scytonemin biosynthesis solely on the basis of comparative genomics was also upregulated. Furthermore, we demonstrate that all of the genes in the 18-gene region are cotranscribed as part of a single transcriptional unit.

A comparative genomics approach to understanding the biosynthesis of the sunscreen scytonemin in cyanobacteria

BackgroundThe extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis of scytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria.ResultsSix putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly of scytonemin from central metabolites, based on genetic, biochemical, and sequence similarity evidence. Also in this cluster are redundant copies of genes encoding for aromatic amino acid biosynthetic enzymes. These can theoretically lead to tryptophan and the tyrosine precursor, p-hydroxyphenylpyruvate, (expected biosynthetic precursors of scytonemin) from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found there as well. We identified four other cyanobacterial strains containing orthologues of all of these genes, three of them by database searches (Lyngbya PCC 8106, Anabaena PCC 7120, and Nodularia CCY 9414) and one by targeted sequencing (Chlorogloeopsis sp. strain Cgs-089; CCMEE 5094). Genomic comparisons revealed that most scytonemin-related genes were highly conserved among strains and that two additional conserved clusters, NpF5232 to NpF5236 and a putative two-component regulatory system (NpF1278 and NpF1277), are likely involved in scytonemin biosynthesis and regulation, respectively, on the basis of conservation and location. Since many of the protein product sequences for the newly described genes, including ScyD, ScyE, and ScyF, have export signal domains, while others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell. Basic structural monomer synthesis and initial condensation are most likely cytoplasmic, while later reactions are predicted to be periplasmic.ConclusionWe show that scytonemin biosynthetic genes are highly conserved among evolutionarily diverse strains, likely include more genes than previously determined, and are predicted to involve compartmentalization of the biosynthetic pathway in the cell, an unusual trait for prokaryotes.

The Global Response of Nostoc punctiforme ATCC 29133 to UVA Stress, Assessed in a Temporal DNA Microarray Study

Cyanobacteria in nature are exposed not only to the visible spectrum of sunlight but also to its harmful ultraviolet components (UVA and UVB). We used Nostoc punctiforme ATCC 29133 as a model to study the UVA response by analyzing global gene expression patterns using genomic microarrays. UVA exposure resulted in the statistically detectable differential expression of 573 genes of the 6903 that were probed, compared with that of the control cultures. Of those genes, 473 were up‐regulated, while only 100 were down‐regulated. Many of the down‐regulated genes were involved in photosynthetic pigment biosynthesis, indicating a significant shift in this metabolism. As expected, we detected the up‐regulation of genes encoding antioxidant enzymes and the sunscreen, scytonemin. However, a majority of the up‐regulated genes, 47%, were unassignable bioinformatically to known functional categories, suggesting that the UVA stress response is not well understood. Interestingly, the most dramatic up‐regulation involved several contiguous genes of unassigned metabolism on plasmid A. This is the first global UVA stress response analysis of any phototrophic microorganism and the differential expression of 8% of the genes of the Nostoc genome indicates that adaptation to UVA in Nostoc has been an evolutionary force of significance.

Role of Colanic Acid Polysaccharide in Serum Resistance In Vivo and in Adherence

Abstract. The production of an extracellular layer of polysaccharide, termed the capsule, is a common feature of many bacteria. Capsules play a vital role in permitting evasion of the host immune specific and nonspecific defenses as well as helping in adhesion for colonization of host tissue. Colanic acid capsule is usually produced in low quantities and is a common feature of several enteric bacteria. The role of the colanic acid capsule in aiding adhesion and virulence was investigated. Encapsulated and unencapsulated cells were injected into granuloma pouches that were formed on the backs of rats. During the first 50 h, the viability of the matched encapsulated and unencapsulated cells decreased. These studies showed that colanic acid capsule does not confer resistance to the bactericidal activity of serum or to phagocytosis in vivo, since there was no significant difference in the survival rates of both strains over time. Adherence studies were conducted in monolayers of human carcinoma intestinal cells (T84) with the same matched strains. After incubating radioactively labeled bacteria with the colon cells, the level of adherence was determined by measuring the radioactivity remaining in the tissue culture wells. The results of these experiments indicated that the unencapsulated cells adhered more readily to the intestinal cells, suggesting that colanic acid capsule interferes with adherence in this model system.

Microbial Growth in a Steady-State Model of Ethylene Glycol-Contaminated Soil

Abstract. Biodegradation of ethylene glycol was tested in a laboratory-scale, steady-state infiltration system of two arid region soil types by monitoring indigenous microbial growth after the infiltration of three concentrations of ethylene glycol. Microorganisms in the soils were able to adapt to the ethylene glycol in several cases, resulting in higher numbers of microorganisms and lower pHs in the effluents. These microorganisms were identified and were able to use ethylene glycol as a sole carbon source. The adaptation was seen best with high-moisture-content soils when the ethylene glycol concentrations were 1% or 10%. However, acclimation to 0.1% and 10% ethylene glycol did not occur in low-moisture-content clay soil, but did occur in low-moisture-content silt soil, indicating that soil type and moisture content are important factors. In all cases, microbial diversity decreased over time.

Cell Surface Display of Poliovirus Receptor on Escherichia coli, a Novel Method for Concentrating Viral Particles in Water

ABSTRACT The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3′ end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.

Optical tracking of a stress-responsive gene amplifier applied to cell-based biosensing and the study of synthetic architectures

A synthetic regulatory construct based on a two-stage amplifying promoter cascade is applied to whole-cell biosensing. Green fluorescent protein (GFP) and red fluorescent protein (RFP) enable two-component tracking of the response event, enabling the system to exhibit increased sensitivity, a lower limit of detection, and a unique optical density-free assessment mode. Specifically, the recA and tac promoters are linked by the LacI repressor in Escherichia coli, where DNA-damage activates the recA promoter and the up-regulation of GFP and LacI proteins. LacI represses the tac promoter, down-regulating the otherwise constitutive mCherry transcription. The response of the construct was compared with two singly tagged, conventional recA promoter-reporter constructs: recA::gfpmut3.1 and recA::mCherry. Using a miniature LED-based flow-through optical detector developed for remote sensing applications, limits of detection for the dual reporter construct reached as low as 0.1 nM MMC. By comparison, single-ended reporters recA::mCherry and recA::gfpmut3.1 achieved best limits of detection of 0.25 nM and 2.0 nM, respectively. An approach to three-component optical analysis, based on a system of detectors with coupled calibration equations enables accurate assessments of the red fluorescence, green fluorescence, and biomass of sensor cell suspensions. The system approach is effective at overcoming interferences caused by optically dense samples and overlapping fluorescence spectra. Such a technique may be useful in studying the biological mechanisms which underlie the synthetic regulatory device of this work and others.