Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein

AbstractThe gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data

Specific Involvement of Pilus Type 2a in Biofilm Formation in Group B Streptococcus

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells

Zn²⁺ Uptake in <i>Streptococcus pyogenes</i>: Characterization of <i>adcA </i>and <i>lmb</i> Null Mutants

<div><p>An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the <i>adcA</i> and <i>lmb</i> genes of <i>Streptococcus pyogenes</i> strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in <i>Streptococcus pneumoniae</i>. Null <i>adcA</i> and <i>lmb</i> mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn²⁺ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to <i>lmb</i>, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null <i>adcA</i> mutant, when expression of <i>lmb</i> is required to compensate for the lack of <i>adcA</i> expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.</p></div

Expression of AdcA, Lmb and HtpA during growth in zinc-depleted medium.

<p>Western blot analysis of total cell extracts from <i>S</i>. <i>pyogenes</i> MGAS5005 wild type, Δ<i>adcA</i> and Δ<i>lmb</i> null mutants grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl₂. (<b>A</b>) Western blot using anti-AdcA specific antibodies. (<b>B</b>) Western blot using anti-Lmb specific antibodies. (<b>C</b>) Western blot using anti-HtpA specific antibodies.</p

Susceptibility to Zn²⁺ starvation.

<p>Susceptibility to Zn²⁺ starvation of wild type, Δ<i>adcA</i> null mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.</p

Bacterial strains, plasmids and primers used in this study.

<p>Bacterial strains, plasmids and primers used in this study.</p

Construction of <i>adcA</i> null mutants.

<p>(<b>A</b>) Schematic representation of the <i>adcA</i> locus in S. <i>pyogenes</i> MGAS5005 wild type and Δ<i>adcA</i> deletion mutants. (<b>B</b>) Map of plasmid pMU1328 used for complementation of the Δ<i>adcA</i> deletion mutants. (<b>C</b>) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent Δ<i>adcA</i> null mutants and a Δ<i>adcA</i> complemented with the pMU1328-<i>adcA</i> construct using anti-AdcA specific antibodies.</p

Inhibition of growth by TPEN is rescued by the addition of Zn²⁺ ions.

<p>(<b>A</b>) Susceptibility to Zn²⁺ starvation of wild type, Δ<i>adcA</i> and Δ<i>lmb</i> null mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (<b>B</b>) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn²⁺ in wild type as well as in Δ<i>adcA</i> and Δ<i>lmb</i> null mutants. Error bars represent the standard deviation.</p