Quantitative High-Throughput Screening Methods Designed for Identification of Bacterial Biocontrol Strains with Antifungal Properties.

Large screens of bacterial strain collections to identify potential biocontrol agents often are time-consuming and costly and fail to provide quantitative results. In this study, we present two quantitative and high-throughput methods to assess the inhibitory capacity of bacterial biocontrol candidates against fungal phytopathogens. One method measures the inhibitory effect of bacterial culture supernatant components on the fungal growth, while the other accounts for direct interaction between growing bacteria and the fungus by cocultivating the two organisms. The antagonistic supernatant method quantifies the culture components' antifungal activity by calculating the cumulative impact of supernatant addition relative to the growth of a nontreated fungal control, while the antagonistic cocultivation method identifies the minimal bacterial cell concentration required to inhibit fungal growth by coinoculating fungal spores with bacterial culture dilution series. Thereby, both methods provide quantitative measures of biocontrol efficiency and allow prominent fungal inhibitors to be distinguished from less effective strains. The combination of the two methods sheds light on the types of inhibition mechanisms and provides the basis for further mode-of-action studies. We demonstrate the efficacy of the methods using Bacillus spp. with different levels of antifungal activities as model antagonists and quantify their inhibitory potencies against classic plant pathogens. IMPORTANCE Fungal phytopathogens are responsible for tremendous agricultural losses on an annual basis. While microbial biocontrol agents represent a promising solution to the problem, there is a growing need for high-throughput methods to evaluate and quantify inhibitory properties of new potential biocontrol agents for agricultural application. In this study, we present two high-throughput and quantitative fungal inhibition methods that are suitable for commercial biocontrol screening.

Cell growth of wall-free L-form bacteria is limited by oxidative damage.

The peptidoglycan (PG) cell wall is a defining feature of the bacterial lineage and an important target for antibiotics, such as ß-lactams and glycopeptides. Nevertheless, many bacteria are capable of switching into a cell-wall-deficient state, called the "L-form" [1-3]. These variants have been classically identified as antibiotic-resistant forms in association with a wide range of infectious diseases [4]. L-forms become completely independent of the normally essential FtsZ cell division machinery [3, 5]. Instead, L-form proliferation is driven by a simple biophysical process based on an increased ratio of surface area to cell volume synthesis [6, 7]. We recently showed that only two genetic changes are needed for the L-form transition in Bacillus subtilis [7]. Class 1 mutations work to generate excess membrane synthesis [7]. Until now, the function of the class 2 mutations was unclear. We now show that these mutations work by counteracting an increase in the cellular levels of reactive oxygen species (ROS) originating from the electron transport pathway, which occurs in wall-deficient cells. Consistent with this, addition of a ROS scavenger or anaerobic culture conditions also worked to promote L-form growth without the class 2 mutations in both Gram-positive B. subtilis and Gram-negative Escherichia coli. Our results suggest that physiological compensation for the metabolic imbalance that occurs when cell wall synthesis is blocked is crucial for L-form proliferation in a wide range of bacteria and also provide new insights into the mode of action of antibiotics that target the bacterial cell wall.

Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins.

Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis.

L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to ß-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

Crucial role for membrane fluidity in proliferation of primitive cells.

The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient "L-form" state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.

The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus.

L-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZ-dependent division machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state. Analysis of the transition process identified an unexpected 'escape' step needed for L-form emergence from the rod. Mutations in two different genes, walR and sepF, contribute to the high frequency of escape: walR, a transcriptional regulator involved in cell wall homeostasis; and sepF, required for accurate and efficient cell division. Time-lapse imaging shows that the mutations act by facilitating the release of the L-form from its walled parent cell but that they act in different ways. The walR mutation renders the activity of the protein partially constitutive, inappropriately upregulating the activity of autolytic enzymes that weaken the cell wall. The sepF mutation probably works by perturbing the formation of a properly constructed division septum, generating a mechanical breach in the wall. The new strain provides a powerful experimental system for studying the genetics and cell biology of L-forms.

Sequential XylS-CTD binding to the Pm promoter induces DNA bending prior to activation.

XylS protein, a member of the AraC family of transcriptional regulators, comprises a C-terminal domain (CTD) involved in DNA binding and an N-terminal domain required for effector binding and protein dimerization. In the absence of benzoate effectors, the N-terminal domain behaves as an intramolecular repressor of the DNA binding domain. To date, the poor solubility properties of the full-length protein have restricted XylS analysis to genetic approaches in vivo. To characterize the molecular consequences of XylS binding to its operator, we used a recombinant XylS-CTD variant devoid of the N-terminal domain. The resulting protein was soluble and monomeric in solution and activated transcription from its cognate promoter in an effector-independent manner. XylS binding sites in the Pm promoter present an intrinsic curvature of 35 degrees centered at position -42 within the proximal site. Gel retardation and DNase footprint analysis showed XylS-CTD binding to Pm occurred sequentially: first a XylS-CTD monomer binds to the proximal site overlapping the RNA polymerase binding sequence to form complex I. This first event increased Pm bending to 50 degrees and was followed by the binding of the second monomer, which further increased the observed global curvature to 98 degrees. This generated a concomitant shift in the bending center to a region centered at position -51 when the two sites were occupied (complex II). We propose a model in which DNA structure and binding sequences strongly influence XylS binding events previous to transcription activation.

Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding.

XylS, an AraC family protein, activates transcription from the benzoate degradation pathway Pm promoter in the presence of a substrate effector such as 3-methylbenzoate (3MB). We developed a procedure to obtain XylS-enriched preparations which proved suitable to analyze its activation mechanism. XylS showed specific 3MB-independent binding to its target operator, which became strictly 3MB dependent in a dimerization-defective mutant. We demonstrated that the N-terminal domain of the protein can make linker-independent interactions with the C-terminal domain and inhibit its capacity to bind DNA. Interactions are hampered in the presence of 3MB effector. We propose two independent roles for 3MB in XylS activation: in addition to its known influence favoring protein dimerization, the effector is able to modify XylS conformation to trigger N-terminal domain intramolecular derepression. We also show that activation by XylS involves RNA polymerase recruitment to the Pm promoter as demonstrated by chromatin immunoprecipitation assays. RNA polymerase switching in Pm transcription was reproduced in in vitro transcription assays. All sigma(32)-, sigma(38)-, and sigma(70)-dependent RNA polymerases were able to carry out Pm transcription in a rigorous XylS-dependent manner, as demonstrated by the formation of open complexes only in the presence of the regulator.

XylS-Pm promoter interactions through two helix-turn-helix motifs: identifying XylS residues important for DNA binding and activation.

The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain, made up of seven alpha-helices conforming two helix-turn-helix DNA-binding domains. alpha-Helix 3 and alpha-helix 6 are the recognition helices. In consonance with XylS structural organization, Pm exhibits a bipartite DNA-binding motif consisting of two boxes, called A and B, whose sequences are TGCA and GGNTA, respectively. This bipartite motif is repeated at the Pm promoter so that one of the XylS monomers binds to each of the repeats. An extensive series of genetic epistasis assays combining mutant Pm promoters and XylS single substitution mutant proteins revealed that alpha-helix 3 contacts A box nucleotides, whereas alpha-helix 6 residues contact B box nucleotides. In alpha-helix 3, Asn246 and Arg242 are involved in specific contacts with the TG dinucleotide at box A, whereas Arg296 and Glu299 contact the second G and T nucleotides at box B. On the basis of our results and of the three-dimensional model of the XylS C-terminal domain, we propose that Ser243, Glu249 and Lys250 in alpha-helix 3, and Asn299 and Arg302 in alpha-helix 6 contact the phosphate backbones. Alanine substitutions at the predicted phosphate backbone-contacting residues yielded mutants with low levels of activity, suggesting that XylS-Pm binding specificity not only involves specific amino acid-base interactions, but also relies on secondary DNA structure, which, although at another level, also comes into play. We propose a model in which a XylS dimer binds to the direct repeats in Pm in a head-to-tail conformation that allows the direct interaction of the XylS proximal subunit with the RNA polymerase sigma factor.

The RpoT regulon of Pseudomonas putida DOT-T1E and its role in stress endurance against solvents.

Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar DeltarpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H(2)O(2), and benzoate. In the DeltarpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the DeltarpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.

Transcriptional tradeoff between metabolic and stress-response programs in Pseudomonas putida KT2440 cells exposed to toluene.

When Pseudomonas putida KT2440 cells encounter toluene in the growth medium, they perceive it simultaneously as a potential nutrient to be metabolized, as a membrane-damaging toxic drug to be extruded, and as a macromolecule-disrupting agent from which to protect proteins. Each of these inputs requires a dedicated transcriptional response that involves a large number of genes. We used DNA array technology to decipher the interplay between these responses in P. putida KT2440 subjected to a short challenge (15 min) with toluene. We then compared the results with those in cells exposed to o-xylene (a non-biodegradable toluene counterpart) and 3-methylbenzoate (a specific substrate of the lower TOL pathway of the P. putida pWW0 plasmid). The resulting expression profiles suggest that the bulk of the available transcriptional machinery is reassigned to endure general stress, whereas only a small share of the available machinery is redirected to the degradation of the aromatic compounds. Specifically, both toluene and o-xylene induce the TOL pathways and a dedicated but not always productive metabolic program. Similarly, 3-methylbenzoate induces the expression not only of the lower meta pathway but also of the non-productive and potentially deleterious genes for the metabolism of (nonsubstituted) benzoate. In addition, toluene (and to a lesser extent o-xylene) inhibit motility functions as an unequivocal response to aromatic toxicity. We argue that toluene is sensed by P. putida KT2440 as a stressor rather than as a nutrient and that the inhibition by the aromatic compounds of many functions we tested is the tradeoff for activating stress tolerance genes at a minimal cost in terms of energy.

RNA polymerase holoenzymes can share a single transcription start site for the Pm promoter. Critical nucleotides in the -7 to -18 region are needed to select between RNA polymerase with sigma38 or sigma32.

The Pm promoter of the benzoate meta-cleavage pathway is transcribed with E sigma32 or E sigma38 according to the growth phase, with an identical transcriptional start site. To investigate sequence determinants in the interaction between either of the two RNA polymerases and Pm, all possible single mutants between positions -7 and -18 were generated, and the activity in the exponential and stationary phases of the resulting mutant promoters was compared. The results precisely delimited a -10 element between positions -7 and -12 (TAGGCT), which defined a promoter sharing nucleotides with both sigma38 and sigma32 consensus. The first two and the last positions of this hexamer were crucial for recognition by both polymerases. Position -10 was the only one specifically recognized by E sigma38, whereas positions -8, -9, and the C-track between positions -14 and -17 were important for specific E sigma32 recognition. Western blots showed that sigma32 was only detectable in the exponential phase, and sigma38 appeared in the early stationary phase. In the rpoH mutant KY1429, sigma38 was already present in the exponential growth phase both free and bound to the RNA polymerase core, in good correlation with the transcription levels found. Pm seems to be optimized for recognition by sigma32 as an initial response to the addition of effector to the medium and allows binding of the adaptable sigma38-dependent RNA polymerase in the stationary phase. XylS is always required for Pm transcription. Therefore, the mechanism that controls Pm expression involves specific nucleotide sequences, the abundance of free and core-bound sigma32 and sigma38 factors during growth, and the presence of the regulator activated by an effector.

XylS activator and RNA polymerase binding sites at the Pm promoter overlap.

Transcription from the TOL plasmid meta-cleavage pathway operon, Pm, depends on the XylS protein being activated by a benzoate effector. The XylS binding sites are two imperfect 5'-TGCAN(6)GGNTA-3' direct repeats located between positions -70/-56 and -49/-35 [González-Pérez et al. (1999) J. Biol. Chem. 274, 2286-2290]. An intrinsic bending of 40 degrees, which is not essential for transcription, is centered at position -43. We have determined the potential overlap between the XylS and RNA polymerase binding sites. The insertion of 2 or more bp between C and T at positions -37 and -36 abolished transcription activation by the wild-type XylS and by XylSS229I, a mutant with increased affinity for the XylS binding sites. In contrast, a 1-bp insertion at -37 was permissible, although when in addition to the 1-bp insertion at -37 the mutant promoter had a point mutation at the XylS binding site (C-47-->T), transcription was abolished with the wild-type XylS protein, but not with XylSS229I. The overlap between the proximal XylS binding site and the -35 region recognized by RNA polymerase at positions -35 and -36 appears to be critical for transcription.