Roni Shapira

Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 105 or 106 colony forming units g-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.

Emergence of Novel Streptococcus iniae Exopolysaccharide-Producing Strains following Vaccination with Nonproducing Strains

ABSTRACT Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.

Immunodetection of the bacteriocin lacticin RM: Analysis of the influence of temperature and tween 80 on its expression and activity

ABSTRACT Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 μg ml−1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 μg ml−1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.

Staphylococcal strains adapted to epigallocathechin gallate (EGCG) show reduced susceptibility to vancomycin, oxacillin and ampicillin, increased heat tolerance, and altered cell morphology

Epigallocathechin gallate (EGCG) possesses many beneficial properties, such as anticarcinogenicity, antiatherogenicity, as well as antioxidant and antibacterial activities. However, the bacterial response to sublethal concentrations of EGCG has not been studied. Here we investigated whether short exposure of staphylococci strains to sublethal doses of EGCG can lead to adaptation and cross-resistance. Two-hour exposure of five strains to 20 microg/ml of EGCG did not affect the growth rate but significantly elevated the resistance towards antibiotics targeting the bacterial cell wall. The magnitude of cross-resistance towards such antibiotics varied with the staphylococci strain, with Staphylococcus aureus Newman exhibiting the highest magnitude of cross-resistance, showing a 2, 4 and 8-fold increase in resistance towards vancomycin, oxacillin and ampicillin respectively. All EGCG-adapted strains were also more heat tolerant than their control counterparts as derived from the Weibull model. Adaptation to EGCG led to a moderate increase in heat resistance of the adapted strains S. epidermis ATCC 12228, S. aureus Newman, and S. aureus ATCC 29213, and an extremely pronounced increase for S. aureus ATCC 6538 and S. aureus RN4220. The shape of the survival curve also varied with the staphylococci strain. Transmission electron microscopy (TEM) analysis revealed suppressed separation of daughter cells in cultures exposed to EGCG, as evidenced by the pseudomulticellular appearance and by more than 2-fold increase in cell wall thickness. These observations raise concerns over the potential of EGCG utilization in therapy in that it may contribute to the development and enhancement of microbial resistance mechanisms.

Polyamines and Ornithine Decarboxylase Activity during Growth and Differentiation in Sclerotium rolfsii

Changes in polyamine content and in the activity of ornithine decarboxylase (ODC) were followed during growth and differentiation of the plant pathogenic fungus Sclerotium rolfsii, in submerged mycelium liquid cultures and on solid agar cultures. Mycelial growth in submerged cultures was characterized by high putrescine content and ODC activity. Growth cessation, resulting from glucose exhaustion in the medium, was accompanied by a sharp decrease in putrescine content and ODC activity. Spermine, the level of which was initially low, was detected in high amounts after all of the glucose was consumed and when the fungus developed the potential for sclerotium formation. A decrease in spermidine, and especially putrescine content, and an increase in spermine content, were observed during the transition from mycelium to mature sclerotia on solid agar medium. Addition of spermine to solid agar medium increased the number of sclerotia by 40%. The changes in the content of the three polyamines were reversed when sclerotia were allowed to germinate. Moreover, α-difluoromethylornithine, the enzyme-activated inhibitor of ODC, greatly inhibited mycelium growth, sclerotium germination and ODC activity, and this inhibition was completely reversed by the addition of putrescine. Cycloheximide delayed sclerotium germination and initially inhibited ODC activity, but ODC inhibition was relieved as soon as sclerotia began to germinate. The data indicate that specific changes in polyamines are linked with two distinct developmental events in S. rolfsii. Mycelial growth and sclerotium germination are positively correlated, and possibly causally linked, with a marked increase in putrescine content and biosynthesis (while spermine cannot be detected). Differentiation (sclerotium formation), however, is accompanied by a major increase in spermine content.

The role of the outer membrane in formaldehyde tolerance in Escherichia coli VU3695 and Halomonas sp. MAC

To investigate the mechanism of formaldehyde tolerance in Gram-negative bacteria, two formaldehyde-tolerant strains, Escherichia coli VU3695 and Halomonas sp. MAC (DSM 7328), and formaldehyde-sensitive revertants obtained by ethidium bromide or novobiocin treatment were studied. The presence of high levels of formaldehyde dehydrogenase activity alone proved insufficient to confer tolerance to high formaldehyde concentrations, as shown by high activity displayed by formaldehyde-sensitive revertants of Halomonas MAC. Moreover, formaldehyde-tolerant strains also proved to be tolerant to high concentrations of acetaldehyde and glutaraldehyde, which are not oxidized by formaldehyde dehydrogenase. Treatment with sublethal concentrations of EDTA rendered the resistant strains highly sensitive to formaldehyde without affecting the activity of formaldehyde dehydrogenase. Comparison of the outer membrane proteins of formaldehyde-resistant strains with those of their sensitive revertants showed the presence of at least one additional high molecular mass protein in the tolerant strains. It is concluded that formaldehyde tolerance in the bacteria studied depends on the composition and structure of the outer membrane.

A pivotal role for the Streptococcus iniae extracellular polysaccharide in triggering proinflammatory cytokines transcription and inducing death in rainbow trout

Streptococcus iniae is a major pathogen of fish, causing considerable economic losses in Israel, the United States and the Far East. Containment of mortalities through vaccination was recently compromised due to the emergence of novel vaccine-escape strains that are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide (EPS) that is released to the medium. In vitro and in vivo data now indicate that the EPS is a major virulence factor, capable of triggering the proinflammatory cytokine machinery and inducing mortality of fish. Streptococcus iniae EPS might therefore be considered to be responsible for sepsis and death just as lipopolysaccharide is for Gram-negative pathogens.

Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM.

The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp. lactis EZ26, is associated with the presence of a 6-kb plasmid, pHU1. The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI-Eco47III fragment. Sequencing analysis of this fragment revealed the presence of six open reading frames (ORFs). Deletion and mutation analyses showed that orfX and orfY are not required for lacticin RM production or immunity, whereas the other ORFs (lacA, lacF, lacG and lacI) are necessary for the bacteriocins production. Transcription analysis indicated that lacA, lacF and lacG are organized in an operon. lacA is probably the lacticin RM structural gene. It putatively encodes a 134-amino acid peptide, and it does not share homology with known bacteriocins. The deduced LacG protein is hydrophobic and consists of six potential trans-membrane helices. lacF encodes a conserved ATP-binding domain homologous to ABC transporters known in bacteriocin immunity systems. LacF and LacG may form an active ABC transporter. Gene-disruption mutations indicated that both are required for immunity against lacticin RM. lacI encodes a small cationic protein, which is required for the production of and immunity to lacticin RM. Protection was obtained only when lacF, lacG and lacI were present together.

Effects of Subinhibitory Concentrations of Menthol on Adaptation, Morphological, and Gene Expression Changes in Enterohemorrhagic Escherichia coli

ABSTRACT Menthol (C10H20O) possesses antibacterial activity; nevertheless, bacterial adaptation to this compound has never been studied. Here we report that precultivation of enterohemorrhagic Escherichia coli (EHEC) strains in increasing subinhibitory (SI) concentrations of menthol significantly elevates (4- to 16-fold) their resistance to menthol. Concomitant morphological alterations included the appearance of mucoid colonies and reduced biofilm production. Scanning electron microscopy (SEM) examination revealed suppressed curli formation in menthol-adapted cells. Expression of the gene cpsB10 (encoding one of the enzymes responsible for colanic acid production) was elevated in response to SI concentrations of menthol in a laboratory E. coli strain, whereas expression in an rcsC null mutant was reduced, implicating a partial role for the Rcs phosphorelay system in mediating the menthol signal. Adaptation to menthol also reduced expression of the locus of enterocyte effacement-encoded regulator (Ler). This reduction, together with reduced curli and biofilm formation and elevated mucoidity, suggests a general reduction in bacterial virulence following adaptation to menthol. Our results thus suggest menthol as a potential lead in the recently emerging alternative strategy of targeting bacterial virulence factors to develop new types of anti-infective agents.

Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus

ABSTRACT We previously found that a short exposure of Staphylococcus aureus to subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG of sigB and vraSR transcription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, an S. aureus 315 vraSR null mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type and sigB null mutant cells to lysostaphin, but this enhancement was much weaker in the vraSR null mutant. Marked upregulation (about 60-fold) of vraR and upregulation of the peptidoglycan biosynthesis-associated genes murA, murF, and pbp2 (2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter of sas016 (encoding a cell wall stress protein of unknown function which is not induced in vraSR null mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.