Identification and Regulation of the N-Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, is known for its ability to catabolize a wide range of plant compounds. This ability is correlated with the presence of specific carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) devoted to scavenging specific carbohydrates. In this study, we demonstrate that there is an X. campestris pv. campestris CUT system involved in the import and catabolism of N-acetylglucosamine (GlcNAc). Expression of genes belonging to this GlcNAc CUT system is under the control of GlcNAc via the LacI family NagR and GntR family NagQ regulators. Analysis of the NagR and NagQ regulons confirmed that GlcNAc utilization involves NagA and NagB-II enzymes responsible for the conversion of GlcNAc-6-phosphate to fructose-6-phosphate. Mutants with mutations in the corresponding genes are sensitive to GlcNAc, as previously reported for Escherichia coli. This GlcNAc sensitivity and analysis of the NagQ and NagR regulons were used to dissect the X. campestris pv. campestris GlcNAc utilization pathway. This analysis revealed specific features, including the fact that uptake of GlcNAc through the inner membrane occurs via a major facilitator superfamily transporter and the fact that this amino sugar is phosphorylated by two proteins belonging to the glucokinase family, NagK-IIA and NagK-IIB. However, NagK-IIA seems to play a more important role in GlcNAc utilization than NagK-IIB under our experimental conditions. The X. campestris pv. campestris GlcNAc NagR regulon includes four genes encoding TonB-dependent active transporters (TBDTs). However, the results of transport experiments suggest that GlcNAc passively diffuses through the bacterial envelope, an observation that calls into question whether GlcNAc is a natural substrate for these TBDTs and consequently is the source of GlcNAc for this nonchitinolytic plant-associated bacterium.Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, produces extracellular plant cell wall-degrading enzymes which contribute to its pathogenicity by facilitating its spread through plant tissues and give the bacterium access to a ready source of nutrients via the carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) (7, 16, 35). The CUT loci are characterized by the presence of genes encoding regulators, degradative enzymes, inner membrane transporters, and outer membrane TonB-dependent transporters (TBDTs), which have been identified as active carbohydrate transporters (7, 33, 44). However, recently, an example of passive diffusion through a TBDT in Caulobacter crescentus was described (17). X. campestris pv. campestris has 72 TBDTs and belongs to a class of bacteria in which TBDTs are overrepresented (7). Our previous study suggested that there are several CUT loci or systems in this bacterium (7).N-Acetylglucosamine (GlcNAc) is an amino sugar that is used for the synthesis of cell surface structures in bacteria and plays an important role in supplying carbon and energy by entering the glycolytic pathway after it is converted into fructose-6-phosphate (fructose-6P) (1, 9). In a recent comparative study of bacterial GlcNAc utilization pathways and regulatory networks, Yang and coworkers identified conserved and distinct features of the GlcNAc utilization pathway in proteobacteria (48). The expression of X. campestris pv. campestris GlcNAc-specific genes was proposed to be controlled by NagR and NagQ regulators belonging to the LacI and GntR families, respectively. In X. campestris pv. campestris strain ATCC 33913, one predicted binding motif specific for NagQ (designated the NagQ box) consists of two imperfect repeats of the TGGTATT sequence separated by 4 bp and is located upstream of the nagQ gene (XCC3414) (Fig. ​(Fig.1A)1A) (48). This gene is part of the nag cluster and is followed by genes encoding the major facilitator superfamily (MFS) inner membrane transporter NagP (XCC3413), the regulator NagR (XCC3412), the GlcN-6P deaminase NagB-II (XCC3411), and the GlcNAc-6P deacetylase NagA (XCC3410) (Fig. ​(Fig.1A).1A). NagR boxes contain the palindromic sequence AATGACARCGYTGTCATT (bold type indicates less highly conserved nucleotides) and are upstream of genes encoding two glucokinase-like NagK-II proteins (XCC2886 [nagK-IIA] and XCC2943 [nagK-IIB]), as well as 5 genes encoding TBDTs (XCC0531, XCC2887, XCC3045, XCC3408, and XCC2944 located downstream of XCC2943) (Fig. ​(Fig.1A).1A). All of the X. campestris pv. campestris genes located downstream of NagR or NagQ boxes were proposed to belong to a GlcNAc utilization pathway involved in uptake of GlcNAc through the bacterial envelope and subsequent phosphorylation, deacetylation, and deamination, which finally leads to the common metabolic intermediate fructose-6-phosphate (Fig. ​(Fig.1B)1B) (48). It was recently demonstrated that in C. crescentus the TBDT CC0446 gene, which is clustered with other nag genes, is responsible for the uptake of GlcNAc (17). The presence of TBDTs in the GlcNAc regulon, which has been observed in Alteromonadales and Xanthomonadales (48), suggests that genes belonging to the GlcNAc utilization pathway define a new CUT system.Open in a separate windowFIG. 1.X. campestris pv. campestris N-acetylglucosamine (GlcNAc) utilization pathway. (A) Organization of genes in the proposed GlcNAc utilization pathway. NagR boxes are indicated by filled circles, and the NagQ box is indicated by an open circle. (B) GlcNAc is proposed to be transported through the outer membrane by TBDTs and then transported across the inner membrane by the MFS transporter NagP. GlcNAc would then be phosphorylated by nagK-II-encoded enzymes. Subsequent metabolism via the nagA-encoded (GlcNAc-6P deacetylase) and nagB-II-encoded (GlcN-6P deaminase) enzymes results in fructose 6-phosphate (Fru-6P) (48). MFS, major facilitator superfamily; PP, periplasm; TBDT, TonB-dependent transporter.Here we describe characterization of the X. campestris pv. campestris GlcNAc utilization pathway and regulatory network, which involves at least the repressors NagR and NagQ. TBDTs are associated with this pathway, confirming the presence of a GlcNAc CUT system in X. campestris pv. campestris. In this bacterium, GlcNAc entry and catabolism imply that novel families containing a GlcNAc inner membrane transporter and GlcNAc kinases are involved.     

The N-Acetylmuramic Acid 6-Phosphate Etherase Gene Promotes Growth and Cell Differentiation of Cyanobacteria under Light-Limiting Conditions

Inactivation of sll0861 in Synechocystis sp. strain PCC 6803 or the homologous gene alr2432 in Anabaena sp. strain PCC 7120 had no effect on the growth of these organisms at a light intensity of 30 μmol photons m⁻² s⁻¹ but reduced their growth at a light intensity of 5 or 10 μmol photons m⁻² s⁻¹. In Anabaena, inactivation of the gene also significantly reduced the rate of heterocyst differentiation under low-light conditions. The predicted products of sll0861 and alr2432 and homologs of these genes showed similarity to N-acetylmuramic acid 6-phosphate etherase (MurQ), an enzyme involved in peptidoglycan recycling, in Escherichia coli. E. coli murQ and the cyanobacterial homologs could functionally substitute for each other. We hypothesize that murQ in cyanobacteria promotes low-light adaptation through reutilization of peptidoglycan degradation products.Cyanobacteria are procaryotes that perform oxygenic photosynthesis and have a Gram-negative cell wall structure (7). They are found in oceans, bodies of freshwater, and the soil surface and contribute significantly to global primary productivity (33). In many environments, light often is a limiting factor for their growth.The efficiency of light harvesting and the distribution of excitation energy in photosystems are important in low-light adaptation. In Prochlorococcus marinus, high- and low-light-adapted ecotypes differ in the number of pcb genes that encode light-harvesting antenna proteins (3, 11). In Synechocystis sp. PCC 6803, rpaC, a gene required for the transition state, can promote growth in white light at an intensity of 2 μmol photons m⁻² s⁻¹ (10, 22). On the other hand, reutilization of secreted substances or degradation products may promote growth under light-limiting conditions. For example, low-light conditions can stimulate the uptake of amino acids in the cyanobacterium Planktothrix rubescens (31).Bacteria can break down peptidoglycan (PG) and reutilize the degradation products to synthesize new PG. This process is called PG recycling. In cyanobacteria and other Gram-negative bacteria, PG forms a continuous layer completely surrounding the cell between the cytoplasmic membrane and the outer membrane (12). The net-like layer consists of glycan strands cross-linked by short peptides with GlcNAc-anhydro-N-acetylmuramic acid (anhMurNAc)-l-Ala-d-Glu-meso-diaminopimelic acid-d-Ala as the repeating unit (23). In Escherichia coli, PG is degraded to GlcNAc-anhMurNAc-peptides or GlcNAc-anhMurNAc and peptides in the periplasmic space, and the GlcNAc-anhMurNAc-peptides and GlcNAc-anhMurNAc are then imported into the cytoplasm by the permease AmpG (13). GlcNAc-anhMurNAc-peptides are processed into GlcNAc-anhMurNAc and tripeptides by AmpD (anhydro-N-acetylmuramyl-l-Ala amidase) and LdcA (LD-carboxypeptidase) in the cytoplasm and reutilized (13, 26). PG accounts for about 2% of the cell mass in Gram-negative bacteria. The reutilization of PG degradation products may promote growth under nutrient-limiting conditions. However, so far, no experimental evidence directly supports this hypothesis. For example, inactivation of ampG or other genes involved in PG recycling apparently does not affect the normal growth rate of E. coli (8, 13, 14, 27, 30), except that it results in autolysis during the stationary growth phase in an ldcA mutant (26).Cyanobacteria have a PG structure similar to that of Gram-negative bacteria, except for small differences, such as the thickness, degree of cross-linking, and covalent linkage of the polysaccharide (15, 16). In the present study, we found that a gene that is highly conserved in cyanobacteria has a function similar to that of murQ, a gene involved in reutilization of GlcNAc-anhMurNAc in E. coli. As shown in Fig. ​Fig.1,1, GlcNAc-anhMurNAc is processed into GlcNAc and anhMurNAc by NagZ (β-N-acetylglucosaminidase) (8), and then GlcNAc is phosphorylated by NagK (GlcNAc kinase), producing GlcNAc-6-P (24), while anhMurNAc is phosphorylated by AnmK (anhMurNAc kinase), producing MurNAc-6-P (28), and is converted by MurQ (MurNAc-6-P etherase) into GlcNAc-6-P (14, 29). GlcNAc-6-P deacetylase (NagA) further converts GlcNAc-6-P to GlcN-6-P, which can be used in synthesis of new PG or enter carbohydrate metabolism (24). We show here that murQ and its homologs in cyanobacteria can promote growth under light-limiting conditions. Also, in a filamentous N₂-fixing cyanobacterium, Anabaena sp. strain PCC 7120, the murQ homolog enhances heterocyst differentiation at a low light intensity.Open in a separate windowFIG. 1.Schematic diagram showing the PG recycling pathway described by Uehara et al. (29). anhMurNAC, anhydro-N-acetylmuramic acid; GlcN-6-P, glucosamine 6-phosphate; GlcNAc, N-acetylglucosamine; GlcNAc-6-P, N-acetylglucosamine 6-phosphate; MurNAC-6-P, N-acetylmuramic acid 6-phosphate.     

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM⁻ cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.Cytokinesis in Escherichia coli and other gram-negative bacteria proceeds via the coordinated constriction of their envelope layers (outer membrane, inner membrane, and peptidoglycan [PG]) (12, 13, 34, 89). This coordination is achieved by a multi-protein division machine referred to as the septal ring or divisome (20). Assembly of the septal ring begins with the polymerization of the bacterial tubulin protein, FtsZ, into a ring structure just underneath the inner membrane at the prospective site of cell division (8). Once formed, this so-called Z-ring facilitates the recruitment of a number of essential and nonessential division proteins to the division site for the assembly of the trans-envelope divisome organelle (20).A major function of the cytokinetic machinery is to promote the synthesis of the PG layer that will eventually fortify the new poles of the developing daughter cells. PG is a polysaccharide polymer composed of repeating units of N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) linked by a β-1,4-glycosidic bond (46). Attached to the MurNAc sugar is a short peptide that is used to form cross-links between adjacent polysaccharide strands (46). Such cross-links allow for the construction of a cell-shaped PG meshwork that surrounds the cell membrane and protects it from osmotic rupture.A new wave of zonal PG synthesis is initiated at the division site during each cell cycle (23, 25, 72, 77, 91). Several of the major PG synthases called penicillin-binding proteins are components of the divisome organelle and play important roles in the synthesis of PG during division (7, 21, 62, 67, 73, 74, 80, 81, 88, 90). The septal PG layer produced by these and perhaps other components of the divisome is thought to be initially shared by the daughter cells (46). In gram-positive bacteria, this septal PG layer is typically split some time after the daughter cells have been compartmentalized by membrane fusion (11). In gram-negative bacteria, however, the septal PG layer is split shortly after it is formed to allow constriction of the outer membrane to closely follow that of the inner (cytoplasmic) membrane (12, 13, 34, 89). This gives rise to the characteristic constricted appearance of predivisional cells of E. coli and its relatives.PG hydrolysis is required to promote septal PG splitting and eventual daughter cell separation (87). E. coli, like many bacteria, encodes a vast array of factors with known or predicted PG hydrolase activity (at least 30 genes and 11 different protein families) (29, 31, 87). In most cases, the loss of individual PG hydrolase factors has little effect on growth and division, suggesting that there is significant functional overlap between the various hydrolases (87). This dearth of phenotypic information has consequently made it difficult to understand the physiological roles of PG hydrolases and identify the subset of these factors needed for septal PG splitting. An approach that has helped overcome this limitation in E. coli, however, has been the systematic deletion of all members of a particular PG hydrolase family from the genome (22, 44, 45, 63). Thus far, of all the families of PG hydrolases encoded by E. coli, the factors that play the predominant role in cell separation appear to be the LytC-type N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC (44, 45, 69). Loss of all three of these amidases results in a severe defect in cell separation and the formation of extremely long cell chains. This chaining phenotype can be exacerbated by the loss of members of other classes of PG hydrolases like the lytic transglycosylases or d,d-endopeptidases (44, 68). However, relative to strains defective for the amidases, mutants lacking multiple lytic transglycosylases or d,d-endopeptidases alone do not display significant chaining phenotypes in E. coli. These PG hydrolases therefore appear to be playing more of an ancillary role in cell separation.The LytM (lysostaphin/peptidase M23)-domain containing factors (referred to as LytM factors for convenience) are a widely distributed class of putative PG hydrolases that have been poorly characterized with regard to their role in PG biogenesis in E. coli and other bacteria (31). The most well-studied members of this family of factors, LytM and lysostaphin, are metallo-endopeptidases that cleave the pentaglycine cross-bridges found in staphylococcal PG (9, 30, 64). Based on this activity, other LytM factors are also likely to be PG hydrolases but with altered cleavage specificity because pentaglycine cross-bridges are only found among the staphylococci (75). Indeed, the LytM protein, gp13, from the Bacillus subtilis phage Φ29 was recently shown to be a d,d-endopeptidase that cleaves the meso-diaminopimelic acid-d-Ala cross-links of B. subtilis PG (17).E. coli encodes four factors with identifiable LytM-domains: EnvC, NlpD, YgeR, and YebA (29) (Fig. ​(Fig.1).1). Of the four, only EnvC has been studied in appreciable detail. EnvC⁻ mutants have a mild cell separation (chaining) defect when grown in medium containing salt and a severe division defect when grown at high temperatures in medium lacking salt (5, 42, 48, 71). In addition, purified EnvC protein was found to possess PG hydrolase activity using a gel-based zymogram assay, and an EnvC-green fluorescent protein (GFP) fusion exported to the periplasm via the Tat system was shown to be recruited to the division site (5). In all, these results support a model in which EnvC is targeted to the division site to participate directly in septal PG splitting and daughter cell separation.Open in a separate windowFIG. 1.Predicted domain structure of the E. coli LytM factors. Shown is a diagram depicting the predicted domain architecture of the four E. coli factors with identifiable LytM domains. Abbreviations: LytM, LytM domain; LysM, LysM PG-binding domain (29); CC, coiled coil; T, transmembrane domain; SS, signal sequence; SS*, lipoprotein signal sequence. The UniProtKB/Swiss-Prot accession numbers are as follows: EnvC (P37690), NlpD (P0ADA3), YebA (P0AFS9), and YgeR (Q46798).In the present study, we investigated the physiological role(s) of the entire set of E. coli LytM factors by generating mutant strains lacking all possible combinations of them. We found that, like the amidases, LytM factors play a critical role in daughter cell separation. Furthermore, studies of their subcellular localization revealed that NlpD is recruited to the division site along with EnvC, indicating that both of these LytM factors are likely to be participating directly in the septal PG splitting process. We also discovered that mutants lacking multiple LytM factors lyse more slowly and display an altered morphological response relative to wild-type (WT) cells when they are treated with ampicillin. This finding suggests that in addition to cell separation, LytM proteins play a role in the lytic mechanism of β-lactam antibiotics.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.Most bacteria have characteristic cell morphologies maintained during growth (67). The peptidoglycan (PG) component of the cell wall represents in most cases the physical support of various bacterial shapes. PG is a mesh-like polymeric macromolecule which opposes the osmotic pressure of the bacterial cytoplasm and prevents lysis in hypotonic growth environments (29). Isolated PG cell walls (sacculi) retain the shapes of the cells from which they originate while PG disruption causes the formation of osmotically labile spheroplasts, underscoring PG''s essential role in cell shape determination and cellular integrity maintenance. PG is composed of long glycan chains that are oriented roughly along the short axis of rod-shaped Gram-negative bacteria and that are connected by short peptide cross-links (21, 60). Bacterial growth and division necessitate the expansion and division of the PG cell wall, which requires the insertion of new PG material in the preexisting, covalently linked mesh (29). New PG synthesis requires two enzymatic reactions performed by penicillin-binding proteins (PBPs). Glycan chain synthesis is achieved by transglycosylation activity while cross-linkage of glycan chains to the existing mesh is achieved by transpeptidation activity (47). Class A PBPs, called bifunctional or bimodular PBPs (e.g., PBP1a and 1b of Escherichia coli), possess both transpeptidase and transglycosylase domains while class B PBPs, such as PBP2 and PBP3 of E. coli, can perform only transpeptidase reactions (47). Controlled degradation of the PG by cell wall hydrolases is necessary for incorporation of new PG material during growth. Tight coordination between PG synthesis and degradation is required to maintain the integrity of the mesh at all times (29).The bacterial cytoskeleton also plays a central role in cell shape determination and maintenance (7). MreB is a bacterial actin homolog that forms dynamic helical structures underneath the cytoplasmic membrane in most rod-shaped bacteria (8, 34, 37, 56). In some species, the spatial distribution of MreB varies during the cell cycle, changing from a helical/patchy localization pattern throughout the cell to a ring-like distribution near midcell (20, 22, 50, 58). MreB is required for rod shape maintenance as deletion of the MreB-encoding gene or depletion of MreB causes loss of rod shape and cell rounding (20, 22, 34, 63). Other proteins, including MreC, MreD, RodA, PBP2, and RodZ, function along with MreB to maintain rod shape as loss of their function also results in cell rounding (2, 5, 33, 48, 62). Among these rod-morphogenic proteins, only PBP2 has a known enzymatic function, being involved in PG synthesis as an elongation-specific transpeptidase; the others are membrane-spanning or integral membrane proteins (2, 5, 15, 48). The overall involvement of these morphogenetic proteins in rod shape maintenance has led to a model in which they are part of the elongase complex, a PG synthesizing machine that elongates the PG side wall (2, 5, 15, 48, 59). The elongase complex would include PG lytic enzymes and at least one bifunctional PBP required for glycan strand synthesis (15, 59). In Bacillus subtilis, MreB homologs were found to associate with the bifunctional PBP1 (36) and to regulate the localization of the PG hydrolase LytE (9). However, it is still unclear how MreB functions in the context of the proposed elongase complex to determine and maintain rod shape.It has been previously shown that repletion of MreB in lemon-shaped, MreB-depleted Caulobacter crescentus cells leads to the formation of cell filaments that present branches and ectopic stalks (64). To examine how MreB can drive de novo rod shape morphogenesis, we followed a similar strategy except that we used drug treatment to interfere with MreB function. The small molecule 3,4-dichlorobenzyl carbamimidothioate, also known as A22, has been shown to rapidly disrupt MreB localization in vivo and to induce growth-dependent rounding in several Gram-negative bacteria (23, 32, 41, 45, 52). Furthermore, genetic and biochemical experiments have shown that MreB is the direct molecular target of A22 and that A22 binds to MreB''s ATP-binding pocket, inducing a state with low affinity for polymerization (3, 23). As removal of A22 is followed within minutes by recovery of the normal MreB localization pattern (23), this drug represents a convenient tool for rapid and reversible inhibition of MreB function. However, A22 was found to inhibit the growth of an mreB deletion mutant of E. coli, suggesting that it can have MreB-independent toxic effects (35). In this study, we show that the toxicity of A22 varies with the drug concentration, culture medium conditions, and Gram-negative species tested. We identify a similarly potent but less toxic structural analog, MP265 (4-chlorobenzyl carbamimidothioate), as well as nontoxic concentrations and conditions for both A22 and MP265 that induce loss of rod cell morphology in C. crescentus. We also show that recovery of rod shape after drug removal is accompanied by intensive remodeling of PG morphology and composition.     

Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.The Bacillus subtilis spoVB gene was first identified as a locus in which a mutation could produce a block at a late stage of spore development (14, 30). Analysis of this locus revealed that it encoded an apparent integral membrane protein (33), and a detailed analysis of a spoVB null mutant demonstrated a block at a very early step in synthesis of the spore cortex peptidoglycan (PG) (40). The mutant synthesized essentially no cortex and accumulated cytoplasmic PG precursors, the same phenotype found in other mutant strains blocked in functions known to be directly involved in PG polymerization (40). These results suggested that SpoVB plays a direct role in assembly or function of the spore PG synthesis apparatus.PG synthesis is a highly conserved and complex process that must span the cell membrane (reviewed in reference 38). Soluble nucleotide-linked PG precursors are synthesized in the cytoplasm. N-Acetylmuramic acid with a pentapeptide side chain is then transferred to an undecaprenol lipid carrier to produce lipid I, with subsequent addition of N-acetylglucosamine to produce lipid II, undecaprenyl-pyrophosphoryl-N-acetylmuramic acid (pentapeptide)-N-acetylglucosamine. Lipid II is then flipped across the membrane via an unknown mechanism. Two families of proteins have been postulated to perform this function: the SEDS family of integral membrane proteins, including FtsW, RodA, and SpoVE (13), and, more recently, the COG2244 family (23), which includes SpoVB and the MviN (MurJ) protein of Escherichia coli (35). In both cases, loss of a protein within one of these families has been shown to result in a block in PG synthesis and the accumulation of lipid-linked and/or soluble PG precursors (16, 20, 35, 40).In the standard model of PG synthesis, flippase activity brings the disaccharide-pentapeptide moieties to the penicillin-binding proteins (PBPs), which polymerize the PG macromolecule on the outer surface of the membrane (39). The class A, high-molecular-weight PBPs possess an N-terminal glycosyl transferase domain that polymerizes the disaccharides into polysaccharide chains (38). These chains are cross-linked via the transpeptidase activity within the penicillin-binding, C-terminal domains of both the class A and the class B PBPs. The N-terminal domains of the class A PBPs and the closely related monofunctional glycosyl transferases found in some species are the only defined PG glycan strand polymerases, and in several species the presence of at least one of these enzymes is essential. However, in B. subtilis (26) and Enterococcus faecalis (3), strains lacking all of these known glycosyl transferases are viable and produce PG walls, indicating the presence of another glycosyl transferase capable of this activity. This alternate glycosyl transferase is distinct in that it is relatively resistant to moenomycin (3, 26), an inhibitor of the class A PBP glycosyl transferase activity (6).Given the strong and early block in cortex PG polymerization observed to occur in a spoVB mutant (40), we wished to further analyze the potential role of this class of protein. SpoVB is a member of a relatively large family of proteins, COG2244 (23), some of which are involved in polymerization of other polysaccharides in bacteria, archaea, and eukaryotes. Bioinformatic analysis has generally predicted that these proteins span the membrane 12 to 14 times, and in some cases experimental evidence has supported this structure (7, 24). A role generally ascribed to these proteins is the flipping of lipid-linked oligosaccharides, produced on the inner face of a membrane, to the outside, where the oligosaccharides are then further polymerized or transferred to other substrates. Some prominent members of this family include Wzx, which functions in O-antigen synthesis in gram-negative bacteria (41); TuaB, which functions in teichuronic acid synthesis in B. subtilis (36); and Rft1, which functions in protein glycosylation in eukaryotes (12). MviN is essential in some gram-negative species, including Burkholderia pseudomallei, E. coli, and Sinorhizobium meliloti (22, 34), and has been shown to play a role in E. coli PG synthesis (16, 35). A Rhizobium tropici mutation that truncates mviN approximately 50% into the coding sequence was not lethal (29). However, it is not known whether this was the sole mviN homolog in the genome or whether the truncated gene product might be functional.We have analyzed the phenotypic properties of B. subtilis strains lacking other proteins within the COG2244 family that are most closely related to SpoVB. Results suggest that these proteins also play roles in PG synthesis and that, in one case, this role is in a synthetic system that is relatively moenomycin resistant. We postulate that these proteins function in an alternate pathway for PG synthesis that may involve the flipping of lipid-linked PG oligosaccharides rather than lipid II disaccharides.     

Levels of the Secreted Vibrio cholerae Attachment Factor GbpA Are Modulated by Quorum-Sensing-Induced Proteolysis

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae. HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, ΔhapA ΔprtV strains attach to chitin beads more efficiently than either the WT or a ΔhapA ΔprtV ΔgbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.Vibrio cholerae has adapted to lifestyles in dual environments, allowing survival in aquatic locations, as well as the ability to colonize the epithelium of the human small intestine. This intestinal colonization by V. cholerae is a prerequisite for the disease cholera in humans. Intestinal colonization proceeds in a stepwise manner, initiating with attachment to the epithelial cell layer by multiple attachment factors (26). This stable attachment localizes the bacterium in an environment conducive for activation of subsequent virulence factors, including the toxin-coregulated pilus, a type IVb pilus that mediates cell-cell interactions and microcolony formation (27). Cholera toxin (CT) is produced and extracellularly secreted by bacteria within the microcolonies and enters into intestinal epithelial cells. CT causes the disruption of fluid and electrolyte balance and results in the voluminous rice water diarrhea characteristically observed with cholera patients.The ability of V. cholerae to bind to surfaces is crucial for the initial stages of colonization of both the aquatic and intestinal environments. Previous studies observing V. cholerae in the aquatic setting identified the ability of the bacteria to attach to zooplankton and phytoplankton, binding to surface structures that include chitin as a major component (7, 10, 11, 19, 21, 42). Chitin, a polymer consisting primarily of a β-1,4 linkage of GlcNAc monomers, is the most abundant aquatic carbon source and, when presented on the surfaces of zooplankton, aquatic exoskeletons, algae, and plants, provides a substrate for V. cholerae surface binding (8, 19-22). V. cholerae is able to break down chitin into carbon to use as a nutrient source via degradation by secreted chitinases (12). We have described a protein, GbpA (GlcNAc binding protein A), which facilitates the binding of V. cholerae to chitin, specifically to the chitin monomer GlcNAc, a sugar residue that is also found on the surface of epithelial cells (3, 16, 26). GbpA mediates binding to chitin, GlcNAc, and exoskeletons of Daphnia magna, as well as participates in effective intestinal colonization within the infant mouse model of cholera (26). GbpA is a secreted protein that exits the cell via the type 2 secretion system by which it mediates attachment by a yet uncharacterized mechanism (26). Previous studies examining the role of GbpA in binding to surfaces have been conducted utilizing various wild-type (WT) strains of V. cholerae, specifically O395 (26) and N16961 (33). These strains both are of the O1 serogroup but are differentially classified as classical (43) and El Tor biotypes (18), respectively. The classical biotype was responsible for the first six pandemics of cholera, whereas El Tor is the cause of the current pandemic (39).Quorum sensing regulates multiple bacterial processes, including virulence, formation of biofilms, and bioluminescence (25, 35, 36). In contrast to many other bacterial quorum-sensing systems, virulence gene expression and biofilm formation in V. cholerae is expressed under conditions of low cell density and repressed at high cell density (17, 35, 48). HapR, a member of the TetR family of regulatory proteins, is a central regulator on which the three parallel inputs of the V. cholerae quorum-sensing system converge (30, 35). During low-cell-density conditions, characteristic of growth within the aquatic environment or stages of early intestinal colonization, the quorum-sensing system is not engaged. Under conditions of high cell density, bacterial numbers and secreted autoinducer molecules are increased to a level that triggers the V. cholerae quorum-sensing system.HapR regulates gene function in two ways, serving as both an activator and repressor. At high cell density, HapR functions in the capacity of a repressor of the toxin-coregulated pilus and CT virulence cascade (29, 31) as well as a repressor of vps gene expression (17), preventing biofilm formation. In addition to repressing gene expression, at high cell density HapR activates the expression of genes encoding extracellularly secreted proteases HapA and PrtV (14, 17, 23, 45-47). HapA, also referred to as hemagglutinin/protease (HA/P), was first reported as a mucinase by Burnet (6) and later characterized as a zinc- and calcium-dependent metalloprotease (4). Extracellularly secreted via the V. cholerae type 2 secretion pathway (40), HA/P has been demonstrated to cleave fibronectin, lactoferrin, and mucin (15), as well as to participate in the activation of the CT A subunit (5). Further studies have led to the suggestion that HA/P is a detachase, critical for the release of V. cholerae from the surface of intestinal cells (2, 14, 38). PrtV is a second protease encoded by a gene that is activated by HapR (47). It has been demonstrated to be essential for both V. cholerae killing of Caenorhabditis elegans, as well as protecting V. cholerae from predator grazing by various flagellates (32, 45).The data presented here indicate that HapA and PrtV participate in the targeted degradation of the attachment factor GbpA. We demonstrate that GbpA is present during the logarithmic phase of growth and conditions of low cell density but that it is not present in the supernatant of high-cell-density cultures of strains that express functional HapR. Further studies revealed that during stages of high cell density, proteases HapA and PrtV, encoded by HapR-activated genes, are responsible for GbpA degradation in the culture supernatant. These findings suggest that the attachment factor GbpA is potentially a ligand targeted for protease degradation during the epithelial detachment process. This process could aid in the release of V. cholerae back into the aquatic environment following late stages of intestinal colonization.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.DNA replication must be highly coordinated with chromatin assembly and cell division for correct propagation of genetic information and cell survival. Errors arising during DNA replication are corrected through the functions of numerous pathways including checkpoints and a diversity of DNA repair mechanisms (32, 33, 35). However, in the absence of these critical cellular responses, replication errors can lead to the accumulation of mutations and gross chromosomal rearrangements (GCRs) as well as chromosome loss, a condition generally termed genomic instability (33). Genome instability is a hallmark of many cancers as well as other human diseases (24). There are many mechanisms by which GCRs can arise, and over the last few years numerous genes and pathways have been implicated in playing a role in the suppression of GCRs in Saccharomyces cerevisiae and in some cases in the etiology of cancer (27, 28, 33, 39-47, 51, 53, 56, 58, 60), including S. cerevisiae ASF1, which encodes the main subunit of the replication coupling assembly factor (37, 62).Asf1 is involved in the deposition of histones H3 and H4 onto newly synthesized DNA during DNA replication and repair (62), and correspondingly, asf1 mutants are sensitive to chronic treatment with DNA-damaging agents (2, 30, 62). However, asf1 mutants do not appear to be repair defective and can recover from acute treatment with at least some DNA-damaging agents (2, 8, 30, 31, 54), properties similar to those described for rad9 mutants (68). In the absence of Asf1, both the DNA damage and replication checkpoints become activated during normal cell growth, and in the absence of checkpoint execution, there is a further increase in checkpoint activation in asf1 mutants (30, 46, 54). It has been suggested that asf1 mutants are defective for checkpoint shutoff and that this might account for the increased steady-state levels of checkpoint activation seen in asf1 mutants (8); however, another study has shown that asf1 mutants are not defective for checkpoint shutoff and that in fact Asf1 and the chromatin assembly factor I (CAF-I) complex act redundantly or cooperate in checkpoint shutoff (31). Furthermore, Asf1 might be involved in proper activation of the Rad53 checkpoint protein, as Asf1 physically interacts with Rad53 and this interaction is abrogated in response to exogenous DNA damage (15, 26); however, the physiological relevance of this interaction is unclear. Asf1 is also required for K56 acetylation of histone H3 by Rtt109, and both rtt109 mutants and histone H3 variants that cannot be acetylated (38) share many of the properties of asf1 mutants, suggesting that at least some of the requirement for Asf1 in response to DNA damage is mediated through Rtt109 (11, 14, 22, 61). Subsequent studies of checkpoint activation in asf1 mutants have led to the hypothesis that replication coupling assembly factor defects result in destabilization of replication forks which are then recognized by the replication checkpoint and stabilized, suggesting that the destabilized replication forks account for both the increased GCRs and increased checkpoint activation seen in asf1 mutants (30). This hypothesis is supported by other recent studies implicating Asf1 in the processing of stalled replication forks (16, 57). This role appears to be independent of CAF-I, which can cooperate with Asf1 in chromatin assembly (63). Asf1 has also been shown to function in disassembly of chromatin, suggesting other possibilities for the mechanism of action of Asf1 at the replication fork (1, 2, 34). Thus, while Asf1 is thought to be involved in progression of the replication fork, both the mechanism of action and the factors that cooperate with Asf1 in this process remain obscure.Stalled replication forks, particularly those that stall at sites of DNA damage, can be processed by homologous recombination (HR) (6) or by a mechanism known as postreplication repair (PRR) (reviewed in reference 67). There are two PRR pathways, an error-prone pathway involving translesion synthesis (TLS) by lower-fidelity polymerases and an error-free pathway thought to involve template switching (TS) (67). In S. cerevisiae, the PRR pathways are under the control of the RAD6 epistasis group (64). The error-prone pathway depends on monoubiquitination of proliferating cell nuclear antigen (PCNA) on K164 by Rad6 (an E2 ubiquitin-conjugating enzyme) by Rad18 (E3 ubiquitin ligase) (23). This results in replacement of the replicative DNA polymerase with nonessential TLS DNA polymerases, such as REV3/REV7-encoded DNA polymerase ζ (polζ) and RAD30-encoded DNA polη, which can bypass different types of replication-blocking damage (67). The error-free pathway is controlled by Rad5 (E3) and a complex consisting of Ubc13 and Mms2 (E2 and E2 variant, respectively), which add a K63-linked polyubiquitin chain to monoubiquitinated PCNA, leading to TS to the undamaged nascent sister chromatid (4, 25, 65). Furthermore, in addition to modification with ubiquitin, K164 of PCNA can also be sumoylated by Siz1, resulting in subsequent recruitment of the Srs2 helicase and inhibition of deleterious Rad51-dependent recombination events (50, 52, 55), although it is currently unclear if these are competing PCNA modifications or if both can exist on different subunits in the same PCNA trimer. A separate branch of the Rad6 pathway involving the E3 ligase Bre1 monoubiquitinates the histone H2B (29, 69) as well as Swd2 (66), which stimulates Set1-dependent methylation of K4 and Dot1-dependent methylation of K79 of histone H3 (48, 49, 66). Subsequently, K79-methylated H3 recruits Rad9 and activates the Rad53 checkpoint (19, 70). Activation of Rad53 is also bolstered by Rad6-Rad18-dependent ubiquitination of Rad17, which is part of the 9-1-1 complex that functions upstream in the checkpoint pathway (17). Finally, Rad6 complexes with the E3 Ubr1, which mediates protein degradation by the N-end rule pathway (13).Due to the role of the PRR pathways at stalled replication forks and a recent study implicating the Rad6 pathway in the suppression of GCRs (39), we examined the relationship between these ubiquitination and sumoylation pathways and the Asf1 pathway in order to gain additional insights into the function of Asf1 during DNA replication and repair. Our findings suggest that Asf1 has multiple functions that prevent replication damage or act in the cellular responses to replication damage and that these functions are modified by and interact with the PRR pathways. The TLS PRR pathway does not appear to be involved, and both a Dun1-dependent replication checkpoint and HR are important for preventing the deleterious effects of PRR and Asf1 pathway defects. We hypothesize that this newly observed cooperation between Asf1 and the PRR pathways may be required for resolving stalled replication forks, leading to suppression of GCRs and successful DNA replication.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.