The effect of alginate lyase on the gentamicin resistance of Pseudomonas aeruginosa in mucoid biofilms.

AIMS: Pseudomonas aeruginosa can secrete large amounts of alginate during chronic infections and this has been associated with high resistance to antibiotics. The major aim of this study was to investigate whether degradation of extracellular alginate by alginate lyase would increase the sensitivity of Ps. aeruginosa to gentamicin, an aminoglycoside antibiotic. METHODS AND RESULTS: Degradation of alginate from Ps. aeruginosa was monitored using a spectrometric assay. Alginate lyase depolymerized alginate, but calcium and zinc cations at concentrations found in the cystic fibrosis lung reduced enzyme activity. Biofilms formed on agar were partially degraded by alginate lyase, but staining with crystal violet showed that the biomass of biofilms grown in liquid was not significantly affected by the enzyme. Viability testing showed that the sensitivity to gentamicin of biofilm bacteria and of bacteria released from biofilms was unaffected by alginate lyase. CONCLUSIONS: Our results show that at least under the conditions used here alginate lyase does not affect gentamicin resistance of Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study indicates that alginate does not contribute to resistance to gentamicin and so does not provide support for the concept of treating patients with alginate lyase in order to increase the antibiotic sensitivity of Ps. aeruginosa.

Role of lung iron in determining the bacterial and host struggle in cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It is a multiorgan system disease that affects the lungs, gastrointestinal tract, liver, and pancreas. The majority of morbidity and mortality in CF relates to chronic airway infection with a variety of bacterial species, commencing in very early infancy, which results in lung destruction and ultimately organ failure (41, 43). This review focuses on iron homeostasis in the CF lung and its role in determining the success and chronicity of Pseudomonas aeruginosa infection. There have been previous excellent reviews regarding iron metabolism in the lower respiratory tract and mechanisms of P. aeruginosa iron acquisition, and we direct readers to these articles for further background reading (31, 53, 58, 77, 96). In this review, we have brought the "two sides of the coin" together to provide a holistic overview of the relationship between host and bacterial iron homeostasis and put this information into the context of current understanding on infection in the CF lung.

Control of gene expression in the P2-related temperate coliphages. V. The use of sequence analysis of 186 Vir mutants to indicate presumptive repressor binding sites.

The prophage of coliphage 186 produces a repressor protein that is required for maintenance of lysogeny and that renders lysogenic cells immune to superinfection by 186. The repressor is likely to be a DNA-binding protein that prevents transcription of the 186 early-lytic genes from promoter pR. To identify the binding site of the repressor, we have isolated virulent mutants that are able to form plaques in the presence of repressor and determined their DNA sequences around pR. The mutants all have mutations in an inverted repeat within pR, and we predict that this repeat is the primary binding site of the repressor. Many of the mutants have second mutations near pR, which allow them to form plaques in the presence of higher concentrations of repressor. The sequences containing these "secondary" mutations show no homology with the putative repressor-binding site, and the role of these mutations in virulence is not clear.

Construction and use of a self-cloning promoter probe vector for gram-negative bacteria.

Transposon Tn5 has been used extensively for the genetic analysis of Gram- bacteria. We describe here the construction and use of a Tn5 derivative which contains the ColE1 origin of DNA replication, thereby allowing the cloning of DNA adjacent to the Tn without the need for construction of genomic libraries. The Tn is derived from Tn5-B21 [Simon et al., Gene 80 (1989) 161-169] and contains a promoter-probe lacZ gene and genes encoding resistance to tetracycline and beta-lactams. It is housed within a mobilisable suicide plasmid which can be transferred to a wide range of Gram- bacteria. The Tn was tested using pyoverdine siderophore-synthesis genes (pvd) from Pseudomonas aeruginosa. The simple cloning procedure allowed 15.9 kb of pvd-associated DNA to be cloned; in addition, the lacZ reporter gene allowed the transcription of pvd genes to be studied. The bacteria were resistant to carbenicillin only if the Tn (and hence the beta-lactamase-encoding gene) was downstream from an active promoter.

Characterisation of the pvdE gene which is required for pyoverdine synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) strain PAO synthesises a siderophore, pyoverdine (Pvd), when grown under conditions of iron starvation. Pvd consists of a dihydroxyquinoline group attached to an 8-amino-acid-residue peptide. DNA spanning at least 78 kb of the chromosome is required for Pvd synthesis, but to date only three genes involved in this process have been characterised. We report the characterisation of a fourth Pa gene, pvdE, which we show to be required for Pvd synthesis. The deduced amino acid sequence of PvdE indicates that the protein is a member of the ATP-binding-cassette (ABC) family of membrane transporter proteins, and this is the first example of the involvement of an ABC-type protein in siderophore synthesis.

Molecular analysis of changes in Pseudomonas aeruginosa load during treatment of a pulmonary exacerbation in cystic fibrosis.

BACKGROUND: Intravenous antibiotics for pulmonary exacerbations (PEs) of cystic fibrosis (CF) usually target Pseudomonas aeruginosa. Insights into the CF lung microbiome have questioned this approach. We used RT-qPCR to determine whether intravenous antibiotics reduced P. aeruginosa numbers and whether this correlated with improved lung function. We also investigated antibiotic effects on other common respiratory pathogens in CF. METHODS: Sputa were collected from patients when stable and again during a PE. Sputa were expectorated into a RNA preservation buffer for RNA extraction and preparation of cDNA. qPCR was used to enumerate viable P. aeruginosa as well as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus fumigatus. RESULTS: Fifteen CF patients were followed through 21 PEs. A complete set of serial sputum samples was unavailable for two patients (three separate PEs). P. aeruginosa numbers did not increase immediately prior to a PE, but numbers during intravenous antibiotic treatment were reduced ≥4-log in 6/18 and ≥1-log in 4/18 PEs. In 7/18 PEs, P. aeruginosa numbers changed very little with intravenous antibiotics and one patient demonstrated a ≥2-log increase in P. aeruginosa load. H. influenzae and S. pneumoniae were detected in ten and five PEs respectively, but with antibiotic treatment these bacteria rapidly became undetectable in 6/10 and 4/5 PEs, respectively. There was a negative correlation between P. aeruginosa numbers and FEV1 during stable phase (r(s)=0.75, p<0.05), and reductions in P. aeruginosa load with intravenous antibiotic treatment correlated with improved FEV1 (r(s)=0.52, p<0.05). CONCLUSIONS: Exacerbations are not due to increased P. aeruginosa numbers in CF adults. However, lung function improvements correlate with reduced P. aeruginosa burden suggesting that current antibiotic treatment strategies remain appropriate in most patients. Improved understanding of PE characterised by unchanged P. aeruginosa numbers and minimal lung function improvement following treatment may allow better targeted therapies.

Chlamydia trachomatis serovar differentiation by direct sequence analysis of the variable segment 4 region of the major outer membrane protein gene.

The polymerase chain reaction method was used to amplify DNA from the fourth variable segment of the gene encoding the major outer membrane protein of Chlamydia trachomatis. Direct sequencing of the amplified DNA from prototype strains confirmed previously identified nucleotide sequence differences that were specific for each serovar. This analysis revealed differences in the DNA sequences of prototype strains C/UW-1 and G/IOL-238 from those of prototype strains C/TW-3 and G/UW-57, sequenced previously. This method was also used to determine the serovar types of C. trachomatis in 125 urogenital specimens from infected patients. The most common serovars were E (38%), F (17%), and G and D (14% each). Serovar D was found significantly more often in specimens from men than in specimens from women (P = 0.004). Conversely, serovar G was found significantly more often in specimens from women than in specimens from men (P = 0.026). Only two serovar G isolates gave sequences identical to that of the prototype strain G/IOL-238, suggesting that this strain may be a serovar variant. Three isolates (D+, G-, and J') gave sequences which have not been reported previously. One isolate had the same sequence as the D- serovar variant. Sequence analysis of amplified DNA reveals subtle differences between C. trachomatis strains and provides a very sensitive method for molecular epidemiological analysis.

The experience of a haematemesis and melaena unit: a review of the first 513 consecutive admissions.

This report is of the results of management in a haematemesis and melaena unit at Prince Henry's Hospital, Melbourne. The unit was established in October, 1972, in response to unpublished data for the decades 1950 to 1959 and 1960 to 1969, which showed a mortality of about 15%. In the 39 months to December, 1975, 513 patients were received into a semi-intensive care setting. The unit staff consisted of a group of four surgeons and four physicians working a weekly roster. Primary care and liaison were the responsibility of the gastroenterology registrar. The basic diagnostic measure taken was the routine early use of fibreoptic duodenoscopy. The unit was set up with an agreed policy of management of the common causes of haematemesis and melaena, and data were prospectively recorded in a form suitable for computer analysis in every case. Of the 513 admissions, 378 were of males and 135 were of females. Forty-five patients died, giving an overall admission mortality of 8-8. There were 143 admissions for bleeding duodenal ulceration, 102 for acute peptic ulceration, 47 for chronic gastriculceration, 43 for oesophageal varices, 33 for Mallory-Weiss syndrome and 45 for less common causes of upper gastrintestinal bleeding. In 100 cases the source of bleeding was not discovered. Of the 143 patients admitted for chronic duodenal ulcer, either patients died, giving a mortality of 5-6%; 72 patients underwent operation, with an operative mortality of 9-7%. Of the 47 admitted with bleeding gastric ulcer, nine died (19-1%), while 26 came to operation; the operative mortality was 26-9%. There were 102 admissions for acute peptic ulceration, with an overall mortality of 11-7% (12 patients); 16 patients came to operation, with an operative mortality of 43-7%. Eleven deaths occurred in the 43 patients admitted for bleeding oesophageal varices (25-6%), with 10 patients coming to operation; the operative mortality was 30-0%. An age of greater than 50 years and shock on admission were the most significant factors for poor prognosis in this group of patients.

DNA homology between siderophore genes from fluorescent pseudomonads.

Many species of pseudomonads produce fluorescent siderophores involved in iron uptake. We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp. B10. There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes. The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P. aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.

Characterization of an ECF sigma factor protein from Pseudomonas aeruginosa.

The PvdS protein is essential for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa. PvdS has some sequence similarity to a family of alternative sigma factor proteins (the ECF [extracytoplasmic factor] family) that direct bacterial RNA polymerases to transcribe genes encoding extracytoplasmic factors. PvdS was purified as a His-tagged protein (hPvdS) and this was used to test the hypothesis that PvdS is a sigma factor protein. The purified protein caused core RNA polymerase from Escherichia coli to bind specifically to the promoters of pyoverdine synthesis genes and enabled transcription from these promoters in vitro. In addition, PvdS was found to co-purify with RNA polymerase from P. aeruginosa, indicating that PvdS associates with RNA polymerase inside the bacteria. These results show that PvdS is a sigma factor protein.

Identification of a new sporulation locus, spoIIIF, in Bacillus subtilis.

We have isolated a mutant of Bacillus subtilis, strain 590, which is blocked at stage III of sporulation. The spo mutation which is carried by this strain is linked to pheA by transformation and defines a previously unidentified locus, spoIIIF. The spoIIIF locus is contiguous with the spoVB locus, in which a mutation causes a block at stage V of sporulation. We also give a detailed genetic map of the pheA region of the chromosome.

Use of temperature-sensitive mutants to study gene expression of two closely linked sporulation loci in Bacillus subtilis.

The spoOB and spoIVF loci are contiguous on the chromosome of Bacillus subtilis, so that genes in these loci may be parts of a single polycistronic operon. Temperature-sensitive strains having mutations in these loci were isolated, and temperature-shift experiments were carried out to investigate expression of the genes. The temperature-sensitive periods of spoOB mutants extended from the beginning of sporulation until the end of the stage II. The temperature-sensitive periods of spoIVF strains were during stage IV of sporulation. Therefore, although the spoOB and spoIVF loci are contiguous on the chromosome it is unlikely that genes in them are parts of a single polycistronic operon.

Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads.

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases.

Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.

Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH.

Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor.

Cells of Pseudomonas aeruginosa secrete a fluorescent yellow-green siderophore, pyoverdine, when grown under iron-deficient conditions. We describe here the cloning and characterization of a gene, pvdS, which is required for this process. The pvdS gene is required for expression from promoters of at least two pyoverdine synthesis genes and can cause expression from these promoters in Escherichia coli, where they are otherwise inactive. Sequencing of pvdS revealed that it is a member of a subfamily of RNA polymerase sigma factors which direct the synthesis of extracellular products by bacteria. The pvdS gene is expressed only in iron-starved bacteria, and in E. coli cells at least, expression is regulated by the Fur repressor protein. We propose that in iron-rich cells of P. aeruginosa, Fur binds to the pvdS promoter and prevents expression of the gene; under conditions of iron starvation, repression is relieved and PvdS is made, reprogramming the cells for pyoverdine synthesis.

Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa.

The alternative sigma factor PvdS is required by Pseudomonas aeruginosa for initiation of transcription from pyoverdine (pvd) promoters. Two divergent PvdS-dependent promoters (pvdE and pvdF) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other pvd promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from pvd promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from pvd promoters and are likely to represent a -35 sequence element for this sigma factor.

UV induction of coliphage 186: prophage induction as an SOS function.

Our results show that UV induction of the 186 prophage depends upon the phage function Tum, with the mutant phenotype of turbid plaques on mitomycin plates and the expression of which is controlled by the host LexA protein. Tum function, encoded near the right-hand end of the coliphage 186 chromosome, is under the control of promoter p95. This promoter is overlapped by a sequence closely related to the consensus sequence of the LexA-binding site. It is proposed that inactivation of LexA after UV irradiation (or by genetic means) leads to prophage induction by permitting expression of Tum which, by unknown means, induces prophage. This mechanism is basically different from that seen with the UV-inducible lambdoid coliphages, which are not regulated by LexA.