Two Plant Bacteria, S. meliloti and Ca. Liberibacter asiaticus, Share Functional znuABC Homologues That Encode for a High Affinity Zinc Uptake System

The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria

The Complete Genome Sequence of ‘Candidatus Liberibacter solanacearum’, the Bacterium Associated with Potato Zebra Chip Disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with ‘Candidatus Liberibacter solanacearum’, a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for ‘Ca. L. solanacearum’. Here we present the sequence of the 1.26 Mbp metagenome of ‘Ca. L. solanacearum’, based on DNA isolated from potato psyllids. The coding inventory of the ‘Ca. L. solanacearum’ genome was analyzed and compared to related Rhizobiaceae to better understand ‘Ca. L. solanacearum’ physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, ‘Ca. L. solanacearum’ is related to ‘Ca. L. asiaticus’, a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to ‘Ca. L. asiaticus’, ‘Ca. L. solanacearum’ probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes

Domains Required for Transcriptional Activation Show Conservation in the Mga Family of Virulence Gene Regulators

Mga, or the multigene regulator of the group A streptococcus (GAS) (Streptococcus pyogenes), is a transcriptional regulator of virulence genes important for colonization and immune evasion. All serotypes of the GAS possess one of two divergent mga alleles (mga-1 or mga-2), and orthologues of Mga have also been identified in other pathogenic streptococci. To date, the only functional motifs established within Mga are two amino-terminal DNA-binding domains (HTH-3 and HTH-4). To uncover novel domains, a random mutagenesis screen using an M6 Mga (mga-1) was undertaken to find mutations leading to a defect in transcriptional activation of the Mga-regulated emm gene. In addition to mutations in the established DNA-binding domains, the screen also revealed mutations in a region conserved among several Mga orthologues. Alanine scanning helped resolve the boundaries of this conserved Mga domain (CMD-1) spanning from residues 10 to 15 of the protein, with the two flanking amino acid residues likely involved in protein stability. Transcriptional reporter analyses demonstrated the importance of CMD-1 for activation of Pemm and autoactivation of Pmga in the serotype M6 Mga. Mutational analyses showed that both CMD-1 and HTH-4 are also necessary for activation of the promoter target Pmrp in a divergent serotype M4 Mga (mga-2), suggesting a conserved functionality. However, in contrast to M6, the M4 Mga mutants did not show a defect in autoregulation. Mutation of similar conserved residues in the Mga-like regulator DmgB from S. dysgalactiae subsp. dysgalactiae showed that CMD-1 and HTH-4 are critical for transcriptional activation in this orthologue, implying that a common mechanism of virulence gene activation may exist for members of the Mga family of regulators

Identification of Residues Responsible for the Defective Virulence Gene Regulator Mga Produced by a Natural Mutant of Streptococcus pyogenes

Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of “gain-of-function” mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen

Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “Candidatus Liberibacter asiaticus”▿

ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “Candidatus Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “Ca. Liberibacter asiaticus” gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “Ca. Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “Ca. Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment

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

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

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