Growth of <i>S. meliloti</i> in zinc repleted media.

<p>Increasing concentrations of ZnSO4, ranging from 0 to 0.5 mM (x-axis), was added to media containing different amounts of EDTA (light grey bars: 0 mM, black speckled bars: 0.1 mM, dark grey bars: 0.2 mM, and white bars: 0.4 mM). The growth of <i>S. meliloti</i> in each media was determined by measuring the absorbance of the culture at 600 nm (OD600) ∼20 hrs post inoculation. Data shown is an average of three independent trials with three replicates grown per trial. Error bars shown represent the standard deviations.</p

Sequence alignment of ZnuA orthologues. Orthologues to ZnuA from <i>S. meliloti</i> and <i>Ca.</i> Liberibacter asiaticus were aligned to ZnuA from <i>E. coli</i> using ClustalW.

<p>Residues that are 100% conserved amongst all orthologues are shown in bold. The three conserved His residues involved in binding zinc are shaded gray.</p

Growth of <i>S. meliloti</i> complemented strains.

<p>Deletion mutants of <i>S. meliloti</i> for <b>A)</b><i>znuA</i> (ΔRZA), <b>B)</b><i>znuB</i> (ΔRZB), or <b>C)</b><i>znuC</i> (ΔRZC) were complemented with the corresponding <i>znu</i> genes from either the wild-type <i>S. meliloti</i> (orange bars), or <i>Ca.</i> Liberibacter asiaticus (blue bars). As controls, the wild-type <i>S. meliloti</i> strain (black bars), the uncomplemented deletion strains (white bars), and those containing the vector control (grey bars) were also grown. Strains were grown in different concentrations of EDTA ranging from 0 to 0.2 mM (x-axis). The absorbance at 600 nm (OD₆₀₀) was measured at the time points indicated. Data shown is an average of three independent replicates with error bars representing the standard deviation.</p

Bioinformatic analysis of the components comprising the Znu gene clusters.

a<p>In accordance with the database at the National Center for Biotechnology Information.</p>b<p>Percentages are based upon a ClustalW alignment between the corresponding protein in <i>E. coli.</i></p

Plasmids and strains used in this study.

<p>Plasmids and strains used in this study.</p

Schematic comparison of the Znu gene cluster from <i>E. coli, S. meliloti</i> and <i>Ca.</i> Liberibacter asiaticus.

<p>Znu homologues have been identified in the two intracellular-plant bacterium, <i>S. meliloti</i> and <i>Ca.</i> Liberibacter asiaticus. The arrangement of <i>znuA, znuB,</i> and <i>znuC</i> genes in each bacterium are shown. A BLAST search of the completed genomes revealed that only the <i>E. coli</i> and <i>S. meliloti</i> gene clusters contain the zinc uptake regulator, <i>zur.</i> In <i>E. coli,</i> the <i>zur</i> gene is located 2.2 Mb downstream of <i>znuA</i> while in <i>S. meliloti</i> it is immediately upstream of <i>znuB</i>. Primers are depicted as small arrows and are colored coded to represent the genes in which they are located. Notations above the primers correspond to the final letters in the primer name and are colored according to the genes that they amplify. Names of primers that were used to cross the junction of a gene are listed in black.</p

Sequence comparison of the Znu proteins from <i>Ca.</i> Liberibacter asiaticus vs. <i>Ca.</i> Liberibacter solanacearum.

a<p>Percentages are based upon a ClustalW alignment between the corresponding proteins.</p

Inhibition of growth by EDTA.

<p>Increasing concentrations of EDTA was added to TY media inoculated with either <i>S. meliloti</i> (black bars) or <i>E. coli</i> (grey bars). The absorbance of the culture at 600 nm (OD600) was measured ∼20 hrs post inoculation. The average of three independent experiments is shown with error bars representing the standard deviation.</p

Growth of <i>E. coli</i> complemented strains.

<p>Strains of <i>E. coli</i> with <b>A)</b><i>znuA,</i><b>B)</b><i>znuB, or</i><b>C)</b><i>znuC</i> insertionally inactivated were complemented with the corresponding <i>znu</i> genes from either <i>S. meliloti</i> (green), <i>E. coli</i> (orange), or <i>Ca.</i> Liberibacter asiaticus (red, blue, and brown) or the vector control (black). Strains were grown both in the absence (solid lines) and presence (dotted lines) of 0.4 mM EDTA. The absorbance at 600 nm (OD₆₀₀) was measured at the time points indicated. Graphs are representative of data from three independent experiments.</p

Wolbachia wsp gene sequence accession numbers used in phylogenetic analysis.

<p>Wolbachia wsp gene sequence accession numbers used in phylogenetic analysis.</p