Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs-motifs I, II, and III-in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS (geminivirus Rep sequence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double-and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae.Geminiviruses are plant viruses with small, circular DNA genomes and twin icosahedral capsids (58). They constitute a large family that is divided into the begomovirus, mastrevirus, curtovirus, and topocuvirus genera based on genome arrangement, insect vector, and host range (68). Geminiviruses amplify their single-stranded (ss) genomes in the nuclei of infected cells using a combination of rolling-circle and recombination-mediated replication (reviewed in references 27, 31, and 35). All geminiviruses encode a conserved protein designated Rep (also known as AL1, AC1, C1, L1, or C1:C2), which mediates initiation of viral replication but does not act as a DNA polymerase (33,42,51). Instead, geminiviruses depend on host replication machinery to copy their genomes (28,31,32).Rep is the only geminivirus protein that is essential for viral replication (18, 56). It is a multifunctional protein that mediates virus-specific recognition of its cognate origin (22) and transcriptional repression (17, 69). Rep initiates and terminates viral DNA synthesis (33, 42, 51) and induces the accumulation of host replication factors in infected cells (48). Rep binds specifically to double-stranded DNA (dsDNA) at a repeated sequence in the 5Ј intergenic region of the viral genome (22, 67), cleaves and ligates DNA within an invariant sequence in a hairpin loop of the plus-strand origin (42, 51), and acts as a DNA helicase to unwind vi...

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.Many bacteria have the ability to release sialic acids from complex glycoproteins and oligosaccharides present in the media or on cell surfaces at sites of colonization or infection. To use the released sialic acids as a rich source of carbon and nitrogen for growth, bacteria must have the ability to transport these compounds into the cell and convert the nine carbon sugars into intermediates that enter the central glycolytic pathways. The utilization of N-acetyl neuraminic acid (NANA), one of the sialic acids, has been well studied in Escherichia coli (36, 37), Haemophilus spp. (1, 35), and Clostridium spp. (38), to name a few.In many microorganisms, the genes for NANA utilization are arranged in an operon that may be regulated by a repressor protein, termed NanR. A comprehensive review of the organization and composition of several prokaryotic operons involved in NANA utilization has been published (36). Many of these operons share common components, including a transport gene for NANA (nanT), a gene encoding an aldolase (nanA) that splits NANA into pyruvate and N-acetyl mannosamine (manNAc), a gene encoding a kinase activity (nanK) that phosphorylates manNAc to form manNAc 6-P and, finally, an epimerase gene (nanE) whose product converts manNAc 6-P to N-acetylglucosamine 6-P (NAG 6-P). NAG 6-P then enters the common pathway of aminosugar utilization (21). For a schematic of the NANA utilization pathway in E. coli, the current paradigm of prokaryotic NANA utilization, see Fig. 7A.Bacteroides fragilis possesses a neuraminidase activity, which can liberate free NANA from complex glycoproteins and oligosaccharides. Go...

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transientreplication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.Geminiviruses constitute a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (77, 82). They infect a broad range of plants and cause devastating crop diseases (57, 63). The family Geminiviridae is classified into four genera, Begomovirus, Curtovirus, Topocuvirus, and Mastrevirus, based on their genome organizations, host ranges, and insect vectors (25,26). The largest genus corresponds to the begomoviruses, which can have bipartite genomes (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV).Geminiviruses replicate through double-stranded DNA (dsDNA) intermediates (34). Begomoviruses encode two proteins involved in viral replication. AL1 (also called AC1, C1, and Rep) is essential for replication (23), while AL3 (also called AC3, C3, and REn) enhances viral DNA accumulation (86). AL1 is a multifunctional protein that mediates the virusspecific recognition of its cognate origin (28), is required for the initiation and termination of viral DNA synthesis (28,49,70), and acts as a DNA helicase (18,19). A variety of protein interactions have been demonstrated for TGMV AL1 and other geminivirus replication proteins, including the formation of homomultimers (72) and interactions with AL3/REn (83, 84) a...

Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage leaf curl virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions.

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.