The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.     

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.     

The inhibitory effect of the fungal toxin, destruxin A, on behavioural fever in the desert locust

During an infection locusts behaviourally fever by seeking out higher environmental temperatures. This behaviour places the pathogen at sub-optimal growth temperatures while improving the efficiency of the immune system, thereby prolonging the lifespan of the host. It is therefore in the interest of the pathogen to either adapt to fever-like temperatures or to evolve mechanisms to interfere with, or inhibit fever. We investigated the behavioural fever response of desert locusts to two fungal pathogens. A prolonged fever was observed in locusts infected with Metarhizium acridum. However, fever was comparatively short-lived during infection with Metarhizium robertsii. In both cases restriction of thermoregulation reduced lifespan. Destruxin A (dtx A) produced by M. robertsii, but not M. acridum has previously been associated with the inhibition of the insect immune system. Injection of dtx A during infection with the fever-causing M. acridum inhibited fever and was particularly effective when administered early on in infection. Furthermore, locusts injected with dtx A were more susceptible to M. acridum infection. Therefore engineering M. acridum isolates currently used for locust biocontrol, to express dtx A may improve efficiency of control by interfering with fever.     

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard®. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24 h at 100% relative humidity, 6-day (25 °C) median lethal concentrations (LC₅₀s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm², respectively. The respective LC₅₀s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm². Use of LC₅₀ versus median lethal concentration ratios (comparing LC₅₀s of each isolate to a “standard” strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC₅₀ values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.     

Use of Scorpion Neurotoxins to Improve the Insecticidal Activity of Rachiplusia ou Multicapsid Nucleopolyhedrovirus

The nucleopolyhedrovirus of the mint looper, Rachiplusia ou (RoMNPV, also known as Anagrapha falcifera multicapsid nucleopolyhedrovirus), has previously been shown to kill the European corn borer, Ostrinia nubilalis, and to protect corn plants against this pest more effectively than the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To further improve the performance of RoMNPV against O. nubilalis, we engineered it to express the insect-selective scorpion neurotoxins AaIT and LqhIT2 using the promoters of the AcMNPV p10 and p6.9 genes. RoMNPV recombinant virus expressing AaIT from the p10 promoter produced a reduced quantity of viral occlusions and polyhedrin protein and failed to occlude virions efficiently. Western blot analysis revealed that the amount of AaIT in hemolymph of recombinant virus-infected O. nubilalis was very low compared to that in the hemolymph of infected Heliothis virescens. In all but one case, insertion of the toxins did not alter the dose of virus required to kill three different hosts. Viruses expressing toxins from the p6.9 promoter killed larvae of O. nubilalis significantly faster than wild-type virus, but no significant difference in speed of kill was observed between AaIT- and LqhIT2-expressing viruses in this host. Expression of LqhIT2 reduced survival times of Helicoverpa zea and H. virescens to a significantly greater extent than expression of AaIT. An AcMNPV recombinant expressing LqhIT2 from the p6.9 promoter killed larvae faster than a recombinant utilizing the p10 promoter for expression, but recombinants using these promoters to drive AaIT expression did not exhibit any significant differences in speed of kill.     

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.     

Systematic Mutagenesis of Genes Encoding Predicted Autotransported Proteins of Burkholderia pseudomallei Identifies Factors Mediating Virulence in Mice,Net Intracellular Replication and a Novel Protein Conferring Serum Resistance

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log₁₀ compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.     

Behavioural changes in Schistocerca gregaria following infection with a fungal pathogen: implications for susceptibility to predation

1. Field observations have indicated that infection of locusts and grasshoppers by the fungal entomopathogen Metarhizium anisopliae var. acridum may result in a substantial increase in the host's susceptibility to predation, before death is caused directly by the disease. 2. Laboratory experiments were conducted to examine how the behaviour of the desert locust Schistocerca gregaria Forskål changes following infection by M. anisopliae var. acridum to explore some potential mechanisms underlying this phenomenon. 3. In the first experiment, which involved monitoring general locust activity in small cages throughout the disease incubation period, infected locusts were observed to increase locomotion and bodily movement from 3 days after infection until death (average survival time of 11 days). There was some evidence of reduced feeding and mating behaviour following infection. 4. In a second experiment, locusts were exposed individually to a simulated predator attack and the initiation and strength of any escape responses were measured. Infected locusts were observed to have a reduced escape capability (both the propensity to escape and the strength of the response). In contrast to the relatively early changes in general activity observed in the first experiment, this was only apparent at the late stages of infection shortly before death. 5. Both an increase in movement and general apparency early in the infection process, and reduced escape capability late on, suggest mechanisms whereby the susceptibility of locusts and grasshoppers to predation might be enhanced following infection with M. anisopliae var. acridum.     

Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein

Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 10⁸ conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 10⁷ conidia/g to 1 × 10⁶ conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 10⁴–10⁵ conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field.     

A Yersinia pestis tat Mutant Is Attenuated in Bubonic and Small-Aerosol Pneumonic Challenge Models of Infection but Not As Attenuated by Intranasal Challenge

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD₅₀ was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.     

Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD₅₀) for the wild-type parent, the LD₅₀ of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassiana-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.     

Role of alternative sigma factor 54 (RpoN) from Vibrio anguillarum M3 in protease secretion, exopolysaccharide production, biofilm formation, and virulence

The sigma factor σ⁵⁴ (RpoN) is an important regulator of bacterial response to environmental stresses. Here, we demonstrate the roles of RpoN in Vibrio anguillarum M3 by comparative investigation of physiological phenotypes and virulence of the wild-type, an rpoN mutant, and an rpoN complemented strain. Disruption of rpoN was found to decrease biofilm formation, production of exopolysaccharides, and production of the metalloproteases EmpA and PrtV. Injection experiments in fish showed that the M3 ΔrpoN mutant was attenuated in virulence when administrated either by intramuscular injection or by immersion challenge. Slower proliferation of the mutant in fish was also observed. Complementation of the mutant strain with rpoN restored some of the phenotypes to wild-type levels. RpoN was involved in regulation of some virulence-associated genes, as shown by real-time quantitative reverse PCR analysis. These results revealed a pleiotropic regulatory role of RpoN in biofilm formation, production of proteases and exopolysaccharides, and virulence in V. anguillarum M3.     

EseD, a Putative T3SS Translocon Component of Edwardsiella tarda, Contributes to Virulence in Fish and is a Candidate for Vaccine Development

Edwardsiella tarda has a type III secretion system (T3SS) essential for pathogenesis. EseD, together with EseB and EseC, has been suggested to form a putative T3SS translocon complex, although its further function is unclear. To investigate the physiological role of EseD, a mutant strain of E. tarda was constructed with an in-frame deletion of the entire eseD gene. One finding was that the ?eseD mutant decreased the secretion levels of EseC and EseB proteins. Additionally, the ?eseD mutant showed attenuated swarming and contact-hemolysis abilities. However, the ?eseD mutant showed increased biofilm formation. Complementation of the mutant strain with eseD restored these phenotypes to those similar to the wild-type strain. Furthermore, infection experiments in fish showed that the ?eseD mutant exhibited slower proliferation and a tenfold decrease in virulence in fish. These results indicate a specific role of EseD in the pathogenesis of E. tarda. Finally, recombinant EseD protein elicited high antibody titers in immunized fish and various levels of protection against lethal challenge with the wild-type strain. These results indicate that EseD protein may be a candidate antigen for development of a subunit vaccine against Edwardsiellosis.     

SitA contributes to the virulence of Klebsiella pneumoniae in a mouse infection model

Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.     

Characterization of Shiga Toxin-Producing Escherichia coli Isolates Associated with Two Multistate Food-Borne Outbreaks That Occurred in 2006

Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx₂ and stx_2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Phenotypic variation and genomic variation in insect virulence traits reveal patterns of intraspecific diversity in a locust-specific fungal pathogen

Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host–pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens.     

FliC,a Flagellin Protein,Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda

Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.     

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.