Bacterial Diversity and Abundance in Shell Biofilms from the Freshwater Snail Pleurocera canaliculatum (Cerithioidea: Pleuroceridae).

Mollusk shells provide a hard substrate for aquatic biofilm colonization. While most work has focused on bivalve shells and grazing, little work has focused on gastropod shells and the microbes growing on them. We sampled biofilms from 14 Pleuroceracanaliculatum and analyzed them using a metagenomic approach. Microbial diversity varied between individuals, and rarefaction suggested that 63 snails would need to be sampled to capture all of the estimated genus-level diversity. Cyanobacteria and species of Novosphingobium and Methylosoma were the most abundant taxa across all shells.

Haemodynamic consequences of recurrent insulin-induced hypoglycaemia.

1. Recurring insulin-induced hypoglycaemia (RIIH) often occurs during the therapeutic management of insulin-dependent diabetes mellitus. Controversy currently exists in the literature as to the ability of insulin and/or hypoglycaemia to promote hypertension. Could insulin and/or hypoglycaemia promote adverse pressor effects? If so, under what conditions and through what mechanism? Thus, the present study was performed to evaluate the hypothesis that RIIH produces hypertension via induction of heme oxygenase (HO)-1, promoting a significant increase in endogenous carbon monoxide (CO). 2. Male Sprague-Dawley rats were treated for 2 weeks with varying doses of insulin (1, 3, 5, 7 and 9 U/kg, s.c.) or vehicle and fed normal rat chow or a zinc diet (1 mmol/L) for 2 weeks. Tail-cuff blood pressure, food intake and blood glucose states were monitored daily. 3. A dose-dependent decrease in blood glucose was observed in insulin-treated rats. Blood pressure was significantly elevated in rats treated with 9 and 7 U/kg insulin compared with those treated with other doses of insulin. However, there was no change in urine output among the groups. Feeding of a high-zinc diet to rats treated with 7 U/kg insulin and treatment with the HO-1 inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG; 20 mg/kg) after insulin injection resulted in a significant reduction in blood pressure compared with 7 U/kg insulin injection alone. In addition, HO-1 protein levels in the heart and kidney and endogenous CO levels were reduced in 7 U/kg insulin-treated rats fed the high-zinc diet compared with those treated with the same dose of insulin alone. 4. The results of the present study demonstrate that RIIH promotes hypertension and that restoration of normal CO levels with a high-zinc diet and ZnDPBG reduces blood pressure.

Heme induction with delta-aminolevulinic Acid stimulates an increase in water and electrolyte excretion.

Purpose. Studies were performed to examine hemodynamic and renal function before and after acute induction of the endogenous CO system with delta-aminolevulinic acid (DALA), which drives HO activity. Methods. In vivo studies were conducted on Inactin-anesthetized male Sprague Dawley rats (250-300 g) either with or without chronic pretreatment with L-NAME (50 mg/Kg, q12 hours x4d). Results. DALA (80 µmol/Kg, IV bolus) administration acutely increased endogenous CO production and HO-1 protein. In untreated and L-NAME-pretreated rats, DALA did not alter BP, GFR, or RBF but increased UF, U(Na)V, and U(K)V (untreated: Δ108.8 ± 0.28%, 172.1 ± 18.4%, and 165.2 ± 45.9%; pretreated: Δ109.4 ± 0.29%, 187.3 ± 26.9%, and 197.2 ± 45.7%). Acute administration of biliverdin (20 mg/kg, IV) and bilirubin (30 mg/kg, IV) to similarly treated animals did not alter UF, U(Na)V, and U(K)V. Conclusion. These results demonstrate that heme oxygenase induction increases urine and electrolyte excretion and suggest a direct tubular action of endogenous carbon monoxide.

Inhibition of heme oxygenase augments tubular sodium reabsorption.

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 µmol/kg ip), a photostable HO inhibitor, was given to control rats and N(G)-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg⁻¹·day⁻¹), 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.

The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis.

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557.

Catabolite repression of Escherichia coli biofilm formation.

Biofilm formation was repressed by glucose in several species of Enterobacteriaceae. In Escherichia coli, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein. A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after approximately 24 h failed to repress and generally activated biofilm formation.

Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli.

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.