Proton-Binding Capacity of Staphylococcus aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity

Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most Gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the Gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.     

Influence of Extracellular Polymeric Substances on the Adsorption of Cadmium onto Three Bacterial Species

In this study, we measured the effect of EPS on Cd and proton adsorption behaviors by measuring the extent of adsorption onto biomass with and without the EPS removed via a cation exchange resin. We conducted both Cd adsorption experiments and potentiometric titrations of biomass using three common bacterial species: one Gram-positive (Bacillus subtilis) and two Gram-negative (Shewanella oneidensis, Pseudomonas putida) species. The Cd adsorption experiments were conducted as a function of metal loading in order to probe whether environmentally-low metal loadings lead to different adsorption mechanisms and roles for EPS than the higher metal loadings of most previous adsorption studies. We suspended each biomass sample in a solution of dissolved Cd in 0.01?M NaClO₄ at metal loadings of 1, 2, 5, and 74?μmol/g. Surface complexation modeling (SCM) was used to determine stability constants for the important Cd-bacteria complexes, and the effect of metal loading on the resulting calculated stability constant values was determined.

In general, the measured bulk Cd adsorption behavior is unaffected by EPS removal. However, our potentiometric titration results suggest that EPS removal does alter the distribution of site types, but not the mass-normalized total site concentration within the biomass. SCM suggests that high affinity sulfhydryl sites control Cd binding under low metal loading conditions for B. subtilis and P. putida, and that sulfhydryl sites are present both on the cells and within the EPS for these species. Conversely, the SCM results suggest that Cd-sulfhydryl binding is un-important on the EPS of S. oneidensis.     

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis.

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.     

Bacteriophage infection in rod-shaped gram-positive bacteria: evidence for a preferential polar route for phage SPP1 entry in Bacillus subtilis

Entry into the host bacterial cell is one of the least understood steps in the life cycle of bacteriophages. The different envelopes of Gram-negative and Gram-positive bacteria, with a fluid outer membrane and exposing a thick peptidoglycan wall to the environment respectively, impose distinct challenges for bacteriophage binding and (re)distribution on the bacterial surface. Here, infection of the Gram-positive rod-shaped bacterium Bacillus subtilis by bacteriophage SPP1 was monitored in space and time. We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB, which is encoded by a putative type VII secretion system gene cluster. YueB was found to concentrate at the cell poles and to display a punctate peripheral distribution along the sidewalls of B. subtilis cells. The kinetics of SPP1 DNA entry and replication were visualized during infection. Most of the infecting phages DNA entered and initiated replication near the cell poles. Altogether, our results reveal that the preferentially polar topology of SPP1 receptors on the surface of the host cell determines the site of phage DNA entry and subsequent replication, which occurs in discrete foci.     

Mechanism of chlorpromazine binding by Gram-positive and Gram-negative bacteria

Chlorpromazine forms charge-transfer complexes with xanthene dyes in bacteria. These complexes permit the differentiation of Gram-positive and Gram-negative bacteria in both light and polarization microscopy. The birefringence induced by the charge-transfer complex might explain the molecular basis of bacterial staining.The charge-transfer complexes formed between chorpromazine and xanthene dyes accumulate in the bacterial cell, mainly inside the bacterial cell wall. The complexes give the cells a color, which depends on the chemical composition of the staining structure, and in particular the polysaccharides of the cell wall in bacteria.Metachromatic granules were seen inside Gram-positive bacteria after chlorpromazine and rose bengal staining. Although the nature of these granules remains unclear, this type of binding may have a role in the inhibition of biochemical processes in the bacterial cells.     

Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.     

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal β-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.     

Interactions of two enantiomers of a designer antimicrobial peptide with structural components of the bacterial cell envelope

Antimicrobial peptides (AMPs) have great potential in treating multi-drug resistant bacterial infections. The antimicrobial activity of d -enantiomers is significantly higher than l -enantiomers and sometimes selectively enhanced against Gram-positive bacteria. Unlike phospholipids in the bacterial plasma membrane, the role of other bacterial cell envelop components is often overlooked in the mode of action of AMPs. In this work, we explored the structural interactions between the main different structural components in Gram-negative/Gram-positive bacteria and the two enantiomers of a designer AMP, GL13K. We observed that both l -GL13K and d -GL13K formed self-assembled amyloid-like nanofibrils when the peptides interacted with lipopolysaccharide and lipoteichoic acid, components of the outer membrane of Gram-negative bacteria and cell wall of Gram-positive bacteria, respectively. Another cell wall component, peptidoglycan, showed strong interactions exclusively with d -GL13K and formed distinct laminar structures. This specific interaction between peptidoglycans and d -GL13K might contribute to the enhanced activity of d -GL13K against Gram-positive bacteria as they have a much thicker peptidoglycan layer than Gram-negative bacteria. A better understanding of the specific role of bacterial cell envelop components in the AMPs mechanism of action can guide the design of more effective Gram-selective AMPs.     

细菌胞壁多糖对水体中低浓度Pb2+和Cd2+的吸附研究

室内模拟研究了长春市伊通河天然水环境中优势细菌胞壁多糖对Pb2+和Cd2+吸附,结果发现胞壁多糖对pb2+和Cd2+的吸附量分别在pH为4.5、5.0时最大;且均分为两个阶段,即当pH＜4.5,对Pb2+的吸附量与pH呈正相关,当pH＞4.5时,对Pb2+的吸附量与pH呈负相关;对Cd2+的吸附量在pH＜5.0时与pH呈正相关,在pH＞5.0时与pH呈负相关.温度对胞壁多糖吸附Pb2+和Cd2+影响不显著;吸附体系在8 h达到吸附平衡.共存Cd2+对胞壁多糖吸附Pb2+影响显著,而共存Pb2+对吸附Cd2+不显著.胞壁多糖对Pb2+和Cd2+吸附过程符合Iangmuir和Freundlich热力学等温方程;胞壁多糖吸附Pb2+和Cd2+的动力学过程分为快速阶段和慢速阶段,其中慢速阶段符合二级吸附速率动力学方程.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Raman spectroscopy of xylitol uptake and metabolism in Gram-positive and Gram-negative bacteria

Visible-wavelength Raman spectroscopy was used to investigate the uptake and metabolism of the five-carbon sugar alcohol xylitol by Gram-positive viridans group streptococcus and the two extensively used strains of Gram-negative Escherichia coli, E. coli C and E. coli K-12. E. coli C, but not E. coli K-12, contains a complete xylitol operon, and the viridans group streptococcus contains an incomplete xylitol operon used to metabolize the xylitol. Raman spectra from xylitol-exposed viridans group streptococcus exhibited significant changes that persisted even in progeny grown from the xylitol-exposed mother cells in a xylitol-free medium for 24 h. This behavior was not observed in the E. coli K-12. In both viridans group streptococcus and the E. coli C derivative HF4714, the metabolic intermediates are stably formed to create an anomaly in bacterial normal survival. The uptake of xylitol by Gram-positive and Gram-negative pathogens occurs even in the presence of other high-calorie sugars, and its stable integration within the bacterial cell wall may discontinue bacterial multiplication. This could be a contributing factor for the known efficacy of xylitol when taken as a prophylactic measure to prevent or reduce occurrences of persistent infection. Specifically, these bacteria are causative agents for several important diseases of children such as pneumonia, otitis media, meningitis, and dental caries. If properly explored, such an inexpensive and harmless sugar-alcohol, alone or used in conjunction with fluoride, would pave the way to an alternative preventive therapy for these childhood diseases when the causative pathogens have become resistant to modern medicines such as antibiotics and vaccine immunotherapy.     

Extracellular gelsolin binds lipoteichoic acid and modulates cellular response to proinflammatory bacterial wall components

The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin's interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160-169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-kappaB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.     

Statistical optimization of the lysis agents for Gram-negative bacterial cells in a microfluidic device

Through statistically designed experiments, lysis agents were optimized to effectively disrupt bacterial cells in a microfluidic device. Most surfactants caused the efficient lysis of Gram-positive microbes, but not of Gram-negative bacteria. A Plackett-Burman design was used to select the components that increase the efficiency of the lysis of the Gram-negative bacteriaEscherichia coli. Using this experimental design, both lysozyme and benzalkonium chloride were shown to significantly increase the cell lysis efficiency, and ATP was extracted in proportion to the lysis efficiency. Benzalkonium chloride affected the cell membrane physically, while lysozyme destroyed the cell wall, and the amount of ATP extracted increased through the synergistic interaction of these two components. The two-factor response-surface design method was used to determine the optimum concentrations of lysozyme and benzalkonium chloride, which were found to be 202 and 99 ppm, respectively. The lysis effect was further verified by microscopic observations in the microchannels. These results indicate that Gram-negative cells can be lysed efficiently in a microfluidic device, thereby allowing the rapid detection of bacterial cells using a bioluminescence-based assay of the released ATP.     

Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.

A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.     

Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

Mammalian peptidoglycan recognition protein binds peptidoglycan with high affinity, is expressed in neutrophils, and inhibits bacterial growth

Peptidoglycan recognition protein (PGRP) is conserved from insects to mammals. In insects, PGRP recognizes bacterial cell wall peptidoglycan (PGN) and activates prophenoloxidase cascade, a part of the insect antimicrobial defense system. Because mammals do not have the prophenoloxidase cascade, its function in mammals is unknown. However, it was suggested that an identical protein (Tag7) was a tumor necrosis factor-like cytokine. Therefore, the aim of this study was to identify the function of PGRP in mammals. Mouse PGRP bound to PGN with fast kinetics and nanomolar affinity (K(d) = 13 nm). The binding was specific for polymeric PGN or Gram-positive bacteria with unmodified PGN, and PGRP did not bind to other cell wall components or Gram-negative bacteria. PGRP mRNA and protein were expressed in neutrophils and bone marrow cells, but not in spleen cells, mononuclear cells, T or B lymphocytes, NK cells, thymocytes, monocytes, and macrophages. PGRP was not a PGN-lytic or a bacteriolytic enzyme, but it inhibited the growth of Gram-positive but not Gram-negative bacteria. PGRP inhibited phagocytosis of Gram-positive bacteria by macrophages, induction of oxidative burst by Gram-positive bacteria in neutrophils, and induction of cytokine production by PGN in macrophages. PGRP had no tumor necrosis factor-like cytotoxicity for mammalian cells, and it was not chemotactic on its own or in combination with PGN. Therefore, mammalian PGRP binds to PGN and Gram-positive bacteria with nanomolar affinity, is expressed in neutrophils, and inhibits growth of bacteria.     

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.     

Virulence mechanisms of Gram-positive plant pathogenic bacteria

Actinobacteria and Firmicutes comprise a group of highly divergent prokaryotes known as Gram-positive bacteria, which are ancestral to Gram-negative bacteria. Comparative genomics is revealing that, though plant virulence genes are frequently located on plasmids or in laterally acquired gene clusters, they are rarely shared with Gram-negative bacterial plant pathogens and among Gram-positive genera. Gram-positive bacterial pathogens utilize a variety of virulence strategies to invade their plant hosts, including the production of phytotoxins to allow intracellular and intercellular replication, production of cytokinins to generate gall tissues for invasion, secretion of proteins to induce cankers and the utilization and manipulation of sap-feeding insects for introduction into the phloem sieve cells. Functional analysis of novel virulence genes utilized by Actinobacteria and Firmicutes is revealing how these ancient prokaryotes manipulate plant, and sometimes insect, metabolic processes for their own benefit.