Four species of marine calcifying algae, the coccolithophores
Calcidiscus leptoporus,
Helicosphaera carteri,
Syracosphaera pulchra and
Umbilicosphaera foliosa were grown in laboratory cultures under ...temperatures varying between 14 and 23 °C, and one species,
C. leptoporus, under varying CO
3
2−, ranging from 105 to 219 μmol/kg. Calcium isotope compositions of the coccoliths resemble in both absolute fractionation and temperature sensitivity previous calibrations of marine calcifying species e.g.
Emiliania huxleyi (coccolithophores) and
Orbulina universa (planktonic foraminifera) as well as inorganically precipitated CaCO
3, but also reveal small species specific differences. In contrast to inorganically precipitated calcite, but similar to
E. huxleyi and
O. universa, the carbonate ion concentration of the medium has no statistically significant influence on the Ca isotope fractionation of
C. leptoporus coccoliths; however, combined data of
E. huxleyi and
C. leptoporus indicate that the observed trends might be related to changes of the calcite saturation state of the medium. Since coccoliths constitute a significant portion of the global oceanic CaCO
3 export production, the Ca isotope fractionation in these biogenic structures is important for defining the isotopic composition of the Ca sink of the ocean, one of the key parameters for modelling changes to the marine Ca budget over time. For the present ocean our results are in general agreement with the previously postulated and applied mean value of the oceanic Ca sink (Δ
sed) of about −
1.3‰, but the observed inter- and intra-species differences point to possible changes in Δ
sed through earth history, due to changing physico-chemical conditions of the ocean and shifts in floral and faunal assemblages.
In this study, a unique mucA mutation (designated mucA56) was introduced, which was characterized by deletion of bases 166-333, encoding MucA56 protein with the deletion of the trans-membrane region, ...which then was proved to be cytoplasmic with phoA-mucA fusion method. PAOmucA56 was constructed with homologous recombination; two PAO1 derivatives PAOmucA22 (PDO300) and PAOmucA56 displayed mucoid phenotype on pseudomonas isolation agar (PIA) agar, but PDO300 produced more alginate than PAOmucA56. Scanning confocal laser microscopy was used to observe the biofilm structures of the three strains during various biofilm development stages. PDO300 developed biofilm with low substratum coverage and high structural heterogeneity, while PAOmucA56 and PAO1 formed uniform biofilm with complete substratum coverage. The proteomes of crude protein extracts of biofilm cells revealed that there are 17 candidate proteins differentially expressed between the two kinds of biofilm, which were proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism and so on. We might conclude that alginate production may affect biofilm architecture, and proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism might play a role in biofilm development alternatively.
Alginate biosynthesis in Pseudomonas aeruginosa is a highly regulated process in which algU and mucA genes are key elements. Mutations in mucA gene determine alginate operon overexpression and ...exopolysaccharide overproduction. In our study, 119 strains of P. aeruginosa were isolated from sputa of 96 cystic fibrosis patients and 84/119 showed nonmucoid phenotype, while 35/119 showed mucoid phenotypes. mucA gene was amplified and sequenced in all strains revealing mutations in 29/35 mucoid strains (82%) and in one non-mucoid strain. 4/29 strains showed mutations never described that generated premature stop and much shorter MucA proteins. In all mutated strains, algU gene expression was analyzed to determine if mutations in mucA, resulting in a strong loss of its protein, could significantly influence its function and subsequently the biosynthetic pathways under algU control. Analysis of algU expression disclosed that the length significantly affects the expression of genes involved in the production of alginate and in the motility and hence survival of P. aeruginosa strains in cystic fibrosis lungs.
In Pseudomonas aeruginosa, conversion to the mucoid phenotype marks the onset of an irreversible state of the infection in Cystic Fibrosis (CF) patients. The main pathway for mucoid conversion is ...mutagenesis of the mucA gene, frequently due to -1 bp deletions in a simple sequence repeat (SSR) of 5 Gs (G(5)-SSR(426)). We have recently observed that this mucA mutation is particularly accentuated in Mismatch Repair System (MRS)-deficient cells grown in vitro. Interestingly, previous reports have shown a high prevalence of hypermutable MRS-deficient strains occurring naturally in CF chronic lung infections. Here, we used mucA as a forward mutation model to systematically evaluate the role of G(5)-SSR(426) in conversion to mucoidy in a MRS-deficient background, with this being the first analysis combining SSR-dependent localized hypermutability and the acquisition of a particular virulence/persistence trait in P. aeruginosa. In this study, mucA alleles were engineered with different contents of G:C SSRs, and tested for their effect on the mucoid conversion frequency and mucA mutational spectra in a mutS-deficient strain of P. aeruginosa. Importantly, deletion of G(5)-SSR(426) severely reduced the emergence frequency of mucoid variants, with no preferential site of mutagenesis within mucA. Moreover, although mutagenesis in mucA was not totally removed, this was no longer the main pathway for mucoid conversion, suggesting that G(5)-SSR(426) biased mutations towards mucA. Mutagenesis in mucA was restored by the addition of a new SSR (C(6)-SSR(431)), and even synergistically increased when G(5)-SSR(426) and C(6)-SSR(431) were present simultaneously, with the mucA mutations being restricted to -1 bp deletions within any of both G:C SSRs. These results confirm a critical role for G(5)-SSR(426) enhancing the mutagenic process of mucA in MRS-deficient cells, and shed light on another mechanism, the SSR- localized hypermutability, contributing to mucoid conversion in P. aeruginosa.
The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme ...stress response system conserved in Gram‐negative bacteria. The system includes an ECF sigma factor, AlgU (σE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucAΔG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU–MucA interactions, although it eliminated MucA–MucB interactions in the yeast two‐hybrid system. The mucAΔG440 truncation of the periplasmic C‐terminal tail of MucA destabilized the molecule resulting in low or undetectable steady‐state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucAΔG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucAΔG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this σ factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB–MucA–AlgU–RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.
Replication through damaged sites in DNA requires in Escherichia coli the SOS stress-inducible DNA polymerase V (UmuC), which is specialized for lesion bypass. Homologs of the umuC gene were found on ...native conjugative plasmids, which often carry multiple antibiotic-resistant genes. MucB is a UmuC homolog present on plasmid R46, and its variant plasmid pKM101 has been introduced into Salmonella strains for use in the Ames test for mutagens. Using a translesion replication assay based on a gapped plasmid carrying a site-specific synthetic abasic site in the single-stranded DNA region, we show that MucB is a DNA polymerase, termed pol RI, which is specialized for lesion bypass. The activity of pol RI requires the plasmid-encoded MucA′protein and the E. coli RecA and single-strand DNA binding proteins. Elimination of any of the proteins from the reaction abolished lesion bypass and polymerase activity. The unprocessed MucA could not substitute for MucA′in the bypass reaction. The presence of a lesion bypass DNA polymerase on a native conjugative plasmid, which has a broad host range specificity and carries multiple antibiotic-resistant genes, raises the possibility that mutagenesis caused by pol RI plays a role in the spreading of antibiotic resistance among bacterial pathogens.
To determine the characterization of mucA gene mutation in clinically isolated Pseudomonas aeruginosa (P.aeruginosa), and the relation between mucA mutation and the mucoid phenotype.
A total of 58 ...strains of P. aeruginosa were collected. Of them, 8 were nonmucoid phenotype and 50 were mucoid phenotype. We detected mucA mutations with PCR-SSCP and sequencing analysis. Alginate was examined by colorimetry.
All strains had mucA mutations (100%), 16 of the 50 (32%) isolates contained mucA mutations that could alter the encoding sequence of amino acids, and the rate in nonmucoid isolates was 0. Fourteen mutation sites were found, 5 of which could alter the encoding sequence of amino acids, and the others were silent mutations. The alginate concentration of mucoid P.aeruginosa was higher than the nonmucoid P. aeruginos (P<0.01). The alginate concentration of the isolates which contained mucA mutations that could alter the encoding sequence of amino acid was higher than the strains only with silent mutations (P<0.01
Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor ...sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.
The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λ CI transcriptional repressors and the UmuD-like ...mutagenesis proteins. Various studies support the hypothesis that LexA and λ CI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 angstrom away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λ CI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme.
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