ubiI,a New Gene in Escherichia coli Coenzyme Q Biosynthesis,Is Involved in Aerobic C5-hydroxylation

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.     

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Exposed to Microcin-Producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

FimZ Is a Molecular Link between Sticking and Swimming in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium produces two types of filamentous appendages on its surface. Fimbriae mediate adherence to tissues and cells via receptor-specific interactions, and flagella are the organelles of motility. These appendages play a role in colonization and dissemination, respectively, from infected surfaces and may be important components of bacterial survival. Increased expression of FimZ in serovar Typhimurium resulted in bacteria which were hyperfimbriated but were nonmotile in soft agar. This lack of motility was associated with down regulation of the flhDC master flagellar operon. Therefore, FimZ represents a molecular connection between flagella and fimbrial formation in serovar Typhimurium, indicating that the synthesis of flagella and fimbriae are oppositely controlled.     

Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Km^r as a selectable marker, the insertion element InsTet^G−1 carries the anhydrotetracycline (atc)-regulated outward-directed P_A promoter so that atc-dependent conditional gene knockouts or knockdowns are generated. The complex formed between the purified hyperactive transposase and InsTet^G−1 was electroporated into Escherichia coli or Salmonella enterica serovar Typhimurium, and mutant pools were collected. We used E. coli strains with either TetR or the reverse variant revTetR^r2, while only TetR was employed in Salmonella. Screening of the InsTet^G−1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants for E. coli and 4 atc-regulatable auxotrophic mutants for Salmonella. We have also screened one Salmonella mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTet^G−1 insertion in the gene pyrE or argA survived this procedure, indicating a reduced intracellular growth rate in J774-A.1 cells. The nature of the mutants and the modes of their regulation are discussed.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.     

Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10(-4) per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10(-6) per cell per generation. Two highly virulent S. Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the Salmonella pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.     

Salmonella enterica Serovar Typhimurium and Escherichia coli Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P ≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20°C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of S. enterica serovar Typhimurium and E. coli in manure-fertilized soil. The number of indigenous E. coli in soil was never significantly lower (P < 0.05) than that of S. enterica serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne S. enterica serovar Typhimurium.     

The Escherichia coli mrsC Gene Is Required for Cell Growth and mRNA Decay

We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis. Originally designated mrsC (mRNA stability), the mrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F. Wang et al., J. Bacteriol. 180:1929–1938, 1998). Strains carrying the thermosensitive mrsC505 allele stopped growing soon after the temperature was shifted to 44°C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44°C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant. In an rne-1 mrsC505 double mutant, the average half-life was 19.8 min. Inactivating mrsC significantly increased the half-lives of the trxA, cat, secG, and kan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of mrsC505 multiple mutants at 44°C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.     

Effects of Nondigestible Oligosaccharides on Salmonella enterica Serovar Typhimurium and Nonpathogenic Escherichia coli in the Pig Small Intestine In Vitro

An in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with either Salmonella or nonpathogenic Escherichia coli strains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenic E. coli and ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease in E. coli associated with the jejunum in the presence of mannose (P < 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers of E. coli in jejunal organ cultures of pigs fed the FOS diet (P < 0.05). There was a reduction, though not a significant one, in the association of Salmonella sp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affect E. coli or Salmonella numbers.     

Resuscitation of Salmonella enterica Serovar Typhimurium and Enterohemorrhagic Escherichia coli from the Viable but Nonculturable State by Heat-Stable Enterobacterial Autoinducer

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the σ^s subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.     

Growth rate toxicity phenotypes and homeostatic supercoil control differentiate Escherichia coli from Salmonella enterica serovar Typhimurium

Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.     

Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a “common good” for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP⁺ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP⁺ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.