Bioinformatic analysis of the <i>slr0977 gene cluster.</i>

<p>a. Not Assigned.</p

Gas chromatographic analysis of OAg components^a.

a<p>Composition of OAg from the indicated strain. Data are presented as the Mol% of each sugar from the total carbohydrate extracted. Sums may not add to 100% due to rounding.</p

EPS and OMP profiles are effected by mutations in Δslr0977, Δslr0982, Δslr1610 or in a triple mutant Δ(slr0977;Δsll0574-5).

<p>(A) EPS as purified from wild-type (WT) or the corresponding mutants by mechanical extraction. EPS from a Δslr1213 strain is known to be deficient in EPS production and was loaded as a control. Samples were analyzed by SDS-PAGE and Alcian Blue staining. Black arrow indicates major EPS band. (B) OMP profile of wild-type or mutant Synechocystis as analyzed by SDS-PAGE and Coomassie brilliant blue staining. Molecular weights of protein standards are indicated for both analyses are indicated by numbers on the left.</p

Export of Extracellular Polysaccharides Modulates Adherence of the Cyanobacterium <i>Synechocystis</i>

<div><p>The field of cyanobacterial biofuel production is advancing rapidly, yet we know little of the basic biology of these organisms outside of their photosynthetic pathways. We aimed to gain a greater understanding of how the cyanobacterium <i>Synechocystis</i> PCC 6803 (<i>Synechocystis</i>, hereafter) modulates its cell surface. Such understanding will allow for the creation of mutants that autoflocculate in a regulated way, thus avoiding energy intensive centrifugation in the creation of biofuels. We constructed mutant strains lacking genes predicted to function in carbohydrate transport or synthesis. Strains with gene deletions of <i>slr0977</i> (predicted to encode a permease component of an ABC transporter), <i>slr0982</i> (predicted to encode an ATP binding component of an ABC transporter) and <i>slr1610</i> (predicted to encode a methyltransferase) demonstrated flocculent phenotypes and increased adherence to glass. Upon bioinformatic inspection, the gene products of <i>slr0977, slr0982,</i> and <i>slr1610</i> appear to function in O-antigen (OAg) transport and synthesis. However, the analysis provided here demonstrated no differences between OAg purified from wild-type and mutants. However, exopolysaccharides (EPS) purified from mutants were altered in composition when compared to wild-type. Our data suggest that there are multiple means to modulate the cell surface of <i>Synechocystis</i> by disrupting different combinations of ABC transporters and/or glycosyl transferases. Further understanding of these mechanisms may allow for the development of industrially and ecologically useful strains of cyanobacteria. Additionally, these data imply that many cyanobacterial gene products may possess as-yet undiscovered functions, and are meritorious of further study.</p></div

Strains and Plasmids.

<p>Strains and Plasmids.</p

Primers used in this study.

<p>Primers used in this study.</p

Adherence of <i>Synechocysis</i> mutants to glass culture vessels.

<p>Quantification was carried out by spectrophotometry of crystal violet staining of attached cells. Strains were grown statically in 2-11 at 30°C with 40 µmol photons m⁻¹ s⁻¹ for 4 days. Data are the combination of at least three biological replicates. Representative data of strains adhering to glass culture vessels (inset). Error bars indicate the standard deviation from the mean.</p

Genetic organization of the <i>slr0977</i> gene cluster.

<p>(A) <i>slr0977</i> and <i>slr0982</i> are predicted to encode a permease and ATPase components of an ATP-transporter, respectively. <i>slr0978-0981</i> encode hypothetical proteins of unknown function. <i>slr1610</i> is a predicted methyltransferase. (B) <i>sll0574</i> and <i>sll0575</i> are predicted to encode a permease and ATPase components of an ATP-transporter, respectively.</p

Adhesins and Host Serum Factors Drive Yop Translocation by <i>Yersinia</i> into Professional Phagocytes during Animal Infection

<div><p><i>Yersinia</i> delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by <i>Yersinia pseudotuberculosis</i> (<i>Yptb</i>) is a multi-factorial process requiring several adhesins and host complement. When WT <i>Yptb</i> or a multiple adhesin mutant strain, <i>ΔailΔinvΔyadA</i>, colonized tissues to comparable levels, <i>ΔailΔinvΔyadA</i> translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by <i>ΔailΔinvΔyadA</i> but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that <i>Yersinia</i> adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, <i>Yersinia</i> uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.</p></div

Adhesin mutants have variable, strain dependent effects on translocation into professional phagocytes.

<p>Splenocytes were infected with the indicated ETEM-expressing strains at an MOI of 1∶1 for (<b>A</b>) 1 h with IP2666, (<b>B</b>) 45 min with IP32953 strains or (<b>C</b>) 45 min with YPIII strains. Professional phagocytes were distinguished by cell-type surface marker staining using flow cytometry. The left Y-axis represents the percentage of each cell type in the spleen (white bars) while the right Y-axis represents the percentage of each cell type present in the Blue⁺ population (grey bars). Experiment was repeated 3–8 times (ND, not determined; *P<0.05, **P<0.01 and ***P<0.001 compared to WT).</p