Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance.

UNLABELLED: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE: Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus. In particular, we show that outer membrane transporters RsaFa and RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

(p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis.

Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated ß-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus' re-entry into the cell cycle after termination of fatty acid starvation.

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication.

The master regulator CtrA oscillates during the Caulobacter cell cycle due to temporally regulated proteolysis and transcription. It is proteolysed during the G1-S transition and reaccumulates in predivisional cells as a result of transcription from two sequentially activated promoters, P1 and P2. CtrA reinforces its own synthesis by directly mediating the activation of P2 concurrently with repression of P1. To explore the role of P1 in cell cycle control, we engineered a mutation into the native ctrA locus that prevents transcription from P1 but not P2. As expected, the ctrA P1 mutant exhibits striking growth, morphological and DNA replication defects. Unexpectedly, we found CtrA and its antagonist SciP, but not DnaA, GcrA or CcrM accumulation to be dramatically reduced in the ctrA P1 mutant. SciP levels closely paralleled CtrA accumulation, suggesting that CtrA acts as a rheostat to modulate SciP abundance. Furthermore, the reappearance of CtrA and CcrM in predivisional cells was delayed in the P1 mutant by 0.125 cell cycle unit in synchronized cultures. High levels of ccrM transcription despite low levels of CtrA and increased transcription of ctrA P2 in the ctrA P1 mutant are two examples of robustness in the cell cycle. Thus, Caulobacter can adjust regulatory pathways to partially compensate for reduced and delayed CtrA accumulation in the ctrA P1 mutant.

Use of bacteria for rapid, pH-neutral, hydrolysis of the model hydrophobic carboxylic acid ester p-nitrophenyl picolinate.

Caulobacter crescentus, Escherichia coli and Bacillus subtilis cultures promote the hydrolysis of the model ester p-nitrophenyl picolinate (PNPP) at neutral pH with high efficiency. Hydrolysis is related to cell concentration, while the interaction of PNPP with both bacterial cells and their extracellular molecules is required for a maximum rate of PNPP hydrolysis in C. crescentus cultures. Furthermore, C. crescentus cultures hydrolyze PNPP at concentrations useful in synthetic chemistry.

Not really a smoker? A study on the prevalence of and attitudes to occasional social smoking in a third level institution in Ireland.

BACKGROUND: Occasional smoking is defined as any smoking occurring on a less than daily basis. Social smoking, i.e. smoking primarily in social contexts, is a sub-group of occasional smoking. Data on occasional cigarette smoking and the subset of social smoking among third level students are limited. OBJECTIVES: (1) To determine prevalence of occasional/social smoking among third level students in an Irish university; (2) to evaluate students' attitudes to occasional/social smoking, including perceived benefits and harm; (3) to explore when students commenced occasional/social smoking, their reasons and continued smoking habits; and (4) to determine any influence of other factors, e.g. alcohol consumption, on occasional/social smoking. METHODS: An anonymous online survey was distributed to undergraduates and postgraduates, using SurveyMonkey. Data were analysed in Microsoft Excel. RESULTS: Of 18,407 students surveyed, 1310 (7.1%) responded;1267 (96.7%) provided adequate data for analysis. Of the 1267 students, 423 (33.4%) self-reported as current smokers of whom 106/1267 (8.4%) self-classified as daily smokers and 317/1267 (25%) as occasional smokers. The 25% of occasional smokers comprised 266/1267 (21%) social smokers and 51/1267 (4%) non-social smokers. Occasional smokers tended to start smoking earlier and think less about quitting than daily smokers. Of 423 current smokers, 386 (97.2%) reported that alcohol increased their smoking habits. CONCLUSION: Prevalence of self-reported occasional smoking among university students was higher than daily smoking. Most occasional smokers primarily smoked in social contexts. All current smokers reported that alcohol increased cigarette intake. Effective intervention campaigns tailored to determinants of occasional/social smoking are needed as part of induction to third level.

msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf.

BACKGROUND: Pathogens tolerate stress conditions that include low pH, oxidative stress, high salt and high temperature in order to survive inside and outside their hosts. Lipopolysaccharide (LPS), which forms the outer-leaflet of the outer membrane in Gram-negative bacteria, acts as a permeability barrier. The lipid A moiety of LPS anchors it to the outer membrane bilayer. The MsbB enzyme myristoylates the lipid A precursor and loss of this enzyme, in Salmonella, is correlated with reduced virulence and severe growth defects that can both be compensated with extragenic suppressor mutations. RESULTS: We report here that msbB (or msbB somA) Salmonella are highly sensitive to physiological CO2 (5%), resulting in a 3-log reduction in plating efficiency. Under these conditions, msbB Salmonella form long filaments, bulge and lyse. These bacteria are also sensitive to acidic pH and high osmolarity. Although CO2 acidifies LB broth media, buffering LB to pH 7.5 did not restore growth of msbB mutants in CO2, indicating that the CO2-induced growth defects are not due to the effect of CO2 on the pH of the media. A transposon insertion in the glucose metabolism gene zwf compensates for the CO2 sensitivity of msbB Salmonella. The msbB zwf mutants grow on agar, or in broth, in the presence of 5% CO2. In addition, msbB zwf strains show improved growth in low pH or high osmolarity media compared to the single msbB mutant. CONCLUSION: These results demonstrate that msbB confers acute sensitivity to CO2, acidic pH, and high osmolarity. Disruption of zwf in msbB mutants restores growth in 5% CO2 and results in improved growth in acidic media or in media with high osmolarity. These results add to a growing list of phenotypes caused by msbB and mutations that suppress specific growth defects.

Isolation and Analysis of Suppressor Mutations in Tumor-Targeted msbB Salmonella.

Tumor-targeted Salmonella offers a promising approach to the delivery of therapeutics for the treatment of cancer. The Salmonella strains used, however, must be stably attenuated in order to provide sufficient safety for administration. Approaches to the generation of attenuated Salmonella strains have included deletion of the msbB gene that is responsible for addition of the terminal myristol group to lipid A. In the absence of myristoylation, lipid A is no longer capable of inducing septic shock, resulting in a significant enhancement in safety. However, msbB Salmonella strains also exhibit an unusual set of additional physiological characteristics, including sensitivities to NaCl, EGTA, deoxycholate, polymyxin, and CO2. Suppressor mutations that compensate for these sensitivities include somA, Suwwan, pmrA (C), and zwf. We describe here methods for isolation of strains with compensatory mutations that suppress these types of sensitivities and techniques for determining their underlying genetic changes and analysis of their effects in murine tumor models.

Versatility of global transcriptional regulators in alpha-Proteobacteria: from essential cell cycle control to ancillary functions.

Recent data indicate that cell cycle transcription in many alpha-Proteobacteria is executed by at least three conserved functional modules in which pairs of antagonistic regulators act jointly, rather than in isolation, to control transcription in S-, G2- or G1-phase. Inactivation of module components often results in pleiotropic defects, ranging from cell death and impaired cell division to fairly benign deficiencies in motility. Expression of module components can follow systemic (cell cycle) or external (nutritional/cell density) cues and may be implemented by auto-regulation, ancillary regulators or other (unknown) mechanisms. Here, we highlight the recent progress in understanding the molecular events and the genetic relationships of the module components in environmental, pathogenic and/or symbiotic alpha-proteobacterial genera. Additionally, we take advantage of the recent genome-wide transcriptional analyses performed in the model alpha-Proteobacterium Caulobacter crescentus to illustrate the complexity of the interactions of the global regulators at selected cell cycle-regulated promoters and we detail the consequences of (mis-)expression when the regulators are absent. This review thus provides the first detailed mechanistic framework for understanding orthologous operational principles acting on cell cycle-regulated promoters in other alpha-Proteobacteria.

Physiological effects of nickel chloride on the freshwater cyanobacterium Synechococcus sp. IU 625.

Harmful algal blooms (HABs) are a serious environmental problem globally. The ability of cyanobacteria, one of the major causative agents of HABs, to grow in heavy metal polluted areas is proving a challenge to environmental restoration initiatives. Some cyanobacteria secrete toxins, such as microcystin, that are potentially dangerous to animals and humans. In this study, the physiology of a cyanobacterium was assessed to nickel chloride exposure. Cell growths were monitored throughout the study with various nickel chloride concentrations (0, 10, 25 or 50 mg/L). Morphological abnormalities were observed with microscopic image analyses. Inductively coupled plasma mass spectrometry (ICP-MS) was carried out to trace the distribution of nickel during the growth period. This study provides insight on potential nickel response mechanisms in freshwater cyanobacteria, which may lead to effective HAB prevention strategy development.

Inhibition of herpes simplex virus type 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate.

Green tea polyphenol epigallocatechin gallate (EGCG) is a strong antioxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG, palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for herpes simplex virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of herpes simplex virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 µM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.

Adaption of Synechococcus sp. IU 625 to growth in the presence of mercuric chloride.

Resistance to heavy metals is important for the survival of bacteria in contaminated environments. In this study, we show that the unicellular cyanobacterial species Synechococcus sp. IU 625 adapts to growth in the presence of mercuric chloride, recovering from pigmentation and morphological defects. Cells accumulate mercury within 2 h of growth and by 3 days, the total mercury concentration is significantly reduced, with all remaining mercury associated with the cells. This suggests that Synechococcus sp. IU 625 can convert mercury to a volatile form.

Temperature-induced activation of freshwater Cyanophage AS-1 prophage.

Synechococcus sp. IU 625 is one of the freshwater cyanobacteria responsible for harmful algal blooms (HAB). Cyanophages can serve as natural control agents and may be responsible for algal bloom prevention and disappearance. Cyanophage AS-1, which infects Synechococcus sp. IU 625 (Anacystis nidulans) and Synechococcus cedrorum, plays an important role in the environment, significantly altering the numbers of its hosts. Since seasonal (temperature-dependent) lytic induction of cyanobacterial prophage has been proposed to affect seawater algal blooms, we investigated if the AS-1 lytic cycle could be induced by a shift to high temperature. Our hypothesis was confirmed, as more phages were released at 35°C than at 24°C, with maximal induction observed with a shift from 24 to 35°C. Furthermore, transmission electron microscopy (TEM) images provide direct evidence of lysogenic to lytic conversion with temperature shift. Thus, temperature is an important inducer for AS-1 conversion from lysogenic to lytic cycle and could have applications in terms of modulating cyanobacterial populations in freshwater aquatic environments. The study gives insight into the effect of climate change on the interaction between cyanophage and cyanobacteria in freshwater ecosystems.

pmrA(Con) confers pmrHFIJKL-dependent EGTA and polymyxin resistance on msbB Salmonella by decorating lipid A with phosphoethanolamine.

Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.

Hot spot for a large deletion in the 18- to 19-centisome region confers a multiple phenotype in Salmonella enterica serovar Typhimurium strain ATCC 14028.

Loss of the Salmonella MsbB enzyme, which catalyzes the incorporation of myristate destined for lipopolysaccharide in the outer membrane, results in a strong phenotype of sensitivity to salt and chelators such as EGTA and greatly diminished endotoxic activity. MsbB- salmonellae mutate extragenically to EGTA-tolerant derivatives at a frequency of 10(-4) per division. One of these derivatives arose from inactivation of somA, which suppresses sensitivity to salt and EGTA. Here we show that a second mode of MsbB- suppression is a RecA-dependent deletion between two IS200 insertion elements present in Salmonella enterica serovar Typhimurium strain ATCC 14028 but not in two other wild-type strains, LT2 and SL1344, which lack one of the IS200 elements. This deletion occurs spontaneously in wild-type and MsbB- strain 14028 salmonellae and accounts for about one-third of all of the spontaneous suppressors of MsbB- in strain 14028. It spans the region corresponding to 17.7 to 19.9 centisomes, which includes somA, on the sequenced map of Salmonella LT2 (136 ORFs in that strain; ATCC 14028 and other strains showed variability in this region). In addition to conferring EGTA resistance correlated with somA, the deletion confers a MacConkey galactose resistance phenotype on MsbB- Salmonella, indicating that at least one additional gene (distinct from somA) within the deletion is responsible for this phenotype. In the wild type, the deletion mutant grows with normal exponential growth rate in Luria broth but is chlorate resistant and does not grow on citrate agar. The deletion strains have lost hydrogen sulfide production, nitrate reductase activity, and gas production from glucose fermentation.