Table 1.
Plasmids and strains used in this study.
Figure 1.
Schematic comparison of the Znu gene cluster from E. coli, S. meliloti and Ca. Liberibacter asiaticus.
Znu homologues have been identified in the two intracellular-plant bacterium, S. meliloti and Ca. Liberibacter asiaticus. The arrangement of znuA, znuB, and znuC genes in each bacterium are shown. A BLAST search of the completed genomes revealed that only the E. coli and S. meliloti gene clusters contain the zinc uptake regulator, zur. In E. coli, the zur gene is located 2.2 Mb downstream of znuA while in S. meliloti it is immediately upstream of znuB. 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.
Figure 2.
Operon structure of the znu genes in S. meliloti1021.
Reverse transcription PCR was performed on total RNA isolated from S. meliloti to determine if the znuB and znuC genes were co-transcribed. Primer sets were designed to amplify fragments either internal to znuA (lanes 2–4), znuB (lanes 5–7),and znuC (lanes 8–10) or a fragment that span the junction between znuB-C (lanes 11–13). Reverse transcriptase was added to the reaction in lanes 2, 5, 8, and 11 (RT+) but omitted from lanes 3, 6, 9, and 12 (RT-) to demonstrate that the RNA was free of any detectable DNA contaminations. Genomic DNA was also amplified with the corresponding primers for use as a positive control (lanes 4, 7, 10, and 13). Experiment shown is representative of data from three independent replicates.
Table 2.
Bioinformatic analysis of the components comprising the Znu gene clusters.
Figure 3.
Growth of E. coli complemented strains.
Strains of E. coli with A) znuA, B) znuB, or C) znuC insertionally inactivated were complemented with the corresponding znu genes from either S. meliloti (green), E. coli (orange), or Ca. 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.
Figure 4.
Increasing concentrations of EDTA was added to TY media inoculated with either S. meliloti (black bars) or E. coli (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.
Figure 5.
Growth of S. meliloti complemented strains.
Deletion mutants of S. meliloti for A) znuA (ΔRZA), B) znuB (ΔRZB), or C) znuC (ΔRZC) were complemented with the corresponding znu genes from either the wild-type S. meliloti (orange bars), or Ca. Liberibacter asiaticus (blue bars). As controls, the wild-type S. meliloti 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.
Figure 6.
Growth of S. meliloti in zinc repleted media.
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 S. meliloti 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.
Figure 7.
Sequence alignment of ZnuA orthologues. Orthologues to ZnuA from S. meliloti and Ca. Liberibacter asiaticus were aligned to ZnuA from E. coli using ClustalW.
Residues that are 100% conserved amongst all orthologues are shown in bold. The three conserved His residues involved in binding zinc are shaded gray.
Table 3.
Sequence comparison of the Znu proteins from Ca. Liberibacter asiaticus vs. Ca. Liberibacter solanacearum.