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The adaptive reactions of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043 under starvation conditions were studied. The main emphasis was given to the peculiarities of stress responses depending on the bacterial population densities. When bacteria were subjected to starvation at high population densities (107-109CFUml-1), their adaptive reactions conformed to the conventional conception of bacterial adaptation related to autolysis of part of the population, specific modification of cell ultrastructure, activation of expression of stress responsive genes and acquiring cross protection against other stress factors. In contrast, at low initial population densities (103-105CFUml-1), as described in our recent work, the cell density increased due to multiple cell division despite the absence of exogenous growth substrate. Here we present data that demonstrate that such unconventional behavior is part of a stress response, which provides increased stress tolerance while retaining virulence. Cell morphology and gene expression in high- and low-cell-density starving Pba cultures were compared. Our investigation demonstrates the existence of alternative adaptive strategies enabling pathogenic bacteria to cope with a variety of stress factors, including starvation, especially necessary when residing outside of their host. © 2013 Institut Pasteur.

© 2019, King Abdulaziz City for Science and Technology. The phytohormone abscisic acid (ABA) plays multiple roles in plant survival and fitness. Significant quantities of ABA are constantly introduced into soil via root exudation, root turnover and incorporation of abscised shoot tissues. In addition, some phytopathogenic fungi synthesize ABA in the course of plant–microbe interactions. The accumulation of soil ABA can inhibit seed germination and root growth but despite this observation, the biochemical pathways of ABA conversion by microorganisms and genetic determinants of the process remain unknown. Here we report on the complete genome sequence of strain P6W, an ABA-utilizing isolate of the genus Novosphingobium. Strain P6W was isolated from the rhizosphere of rice (Oryza sativa L.) seedlings using a selective ABA-supplemented medium. The genome of strain P6W consists of 6,606,532 bp, which includes two chromosomes and two plasmids. It comprises of 5663 protein-coding genes and 80 RNA genes. ANI values calculated based on the analysis of nine previously sequenced genomes of members of the genus Novosphingobium ranged from 77 to 92%, which suggests that strain P6W is potentially a new species of the genus Novosphingobium. Functional annotation of genes in the genome of strain P6W revealed a number genes that could be potentially responsible for ABA degradation.

© 2019 Russian Academy of Agricultural Sciences. All rights reserved. Plant pathogenic pectobacteria (Pectobacterium genus) are well-known all over the world as the causal agents of the cultural plant diseases called soft rots. Rot symptoms are related to the extensive plant tissue maceration due to the production by microorganisms of the plant cell wall degrading enzymes. Most of the pectobacteria-secreted enzymes catalyze the cleavage of homogalacturonan. This polysaccharide, that is a linear homopolymer, consists of galacturonic acid residues and is the most abundant (by mass) pectic polysaccharide of plant cell walls. The knockout of genes of homogalacturonan-degrading enzymes is known to lead to reduced virulence of pectobacteria. In addition, the modification of another pectic compound — rhamnogalacturonan I also occurs in the course of infection process caused by pectobacteria. This compound is a ramified heteropolymer, the backbone of which consists of alternate rhamnose and galacturonic acid residues, and side chains are represented by galactose or arabinose residues. However, the role of pectobacterial enzymes for rhamnogalacturonan I degradation in the development of soft rots has not been previously ascertained. The present study is dedicated to the investigation of the necessity of P. atrosepticum SCRI1043 enzymes degrading rhamnogalacturonan I for a full development of soft rots in the plants infected by pectobacteria. By directed mutagenesis, we have obtained mutant forms of P. atrosepticum SCRI1043 deficient in genes encoding rhamnogalacturonyl hydrolase (genome locus eca3749) that cleaves the backbone of rhamnogalacturonan I, and galactanase (genome locus eca0852) that breaks side chains of this polymer. For the target gene knockout, mutant loci were constructed by overlap-extension PCR. Most of the original gene was replaced by kanamicin-resistance cassette. The obtained construction was ligated into a mobilized suicide vector and the resulting pla

© 2019, Springer Nature B.V. In our study, the rare earth element ytterbium (Yb3+) was demonstrated to affect water exchange in roots of Zea mays seedlings. Herewith, the overall membrane permeability (Pd) increased. The Pd increase was determined by aquaporin activity but not the membrane lipid component since the closure of aquaporin channels due to low intracellular pH abolished the positive effect of Yb3+ on Pd. Additionally, the expression level of aquaporin genes ZmPIP2;2, ZmPIP2;6 and ZmTIP2;2 was increased when plants were grown in the presence of Yb3+. Our results indicate that previously described positive influence of rare earth metals on plant growth and productivity may be mediated (at least partially) by the modification of the plant hydraulic system.

© 2019 Gogoleva et al. Mechanisms of microbial catabolism of phytohormone abscisic acid (ABA) are still unknown. Here, we report the complete genome sequence of ABAutilizing Rhodococcus sp. strain P1Y, isolated from the rice (Oryza sativa L.) rhizosphere. The sequence was obtained using an approach combining Oxford Nanopore Technologies MinION and Illumina MiSeq sequence data.

Herein, for the first time the complexation ability of a homological series of triphenylphosphonium surfactants (TPPB-n) toward DNA decamers has been explored. Formation of lipoplexes was confirmed by alternative techniques, including dynamic light scattering, indicating the occurrence of nanosized complexes (ca. 100-150 nm), and monitoring the charge neutralization of nucleotide phosphate groups and the fluorescence quenching of dye-intercalator ethidium bromide. The complexation efficacy of TPPB-surfactants toward an oligonucleotide (ONu) is compared with that of reference cationic surfactants. Strong effects of the alkyl chain length and the structure of the head group on the surfactant/ONu interaction are revealed, which probably occur via different mechanisms, with electrostatic and hydrophobic forces or intercalation imbedding involved. Phosphonium surfactants are shown to be capable of disordering lipid bilayers, which is supported by a decrease in the temperature of the main phase transition, Tm. This effect enhances with an increase in the alkyl chain length, indicating the integration of TPPB-n with lipid membranes. This markedly differs from the behavior of typical cationic surfactant cetyltrimethylammonium bromide, which induces an increase in the Tm value. It was demonstrated that the cytotoxicity of TPPB-n in terms of the MTT-test on a human cell line 293T nonmonotonically changes within the homological series, with the highest cytotoxicity exhibited by the dodecyl and tetradecyl homologs.

© 2018, Pleiades Publishing, Inc. Modern techniques of next-generation sequencing (NGS) allow obtaining expression profile of all genes and provide an essential basis for characterizing metabolism in the organism of interest on a broad scale. An important condition for obtaining a demonstrative physiological picture using high throughput sequencing data is the availability of the genome sequence and its sufficient annotation for the target organism. However, a list of species with properly annotated genomes is limited. Transcriptome profiling is often performed in the so-called non-model organisms, which are those with unknown or poorly assembled and/or annotated genome sequences. The transcriptomes of non-model organisms are possible to investigate using algorithms of de novo assembly of the transcripts from sequences obtained as the result of RNA sequencing. A physiological interpretation of the data is difficult in this case because of the absence of annotation of the assembled transcripts and their classification by metabolic pathway and functional category. An algorithm for transcriptome profiling in non-model organisms was developed, and a transcriptome analysis was performed for the basidiomycete Lentinus edodes. The algorithm includes open access software and custom scripts and encompasses a complete analysis pipeline from the selection of cDNA reads to the functional classification of differentially expressed genes and the visualization of the results. Based on this algorithm, a comparative transcriptome analysis of the nonpigmented mycelium and brown mycelial mat was performed in L. edodes. The comparison revealed physiological differences between the two morphogenetic stages, including an induction of cell wall biogenesis, intercellular communication, ion transport, and melanization in the brown mycelial mat.

© 2018, Pleiades Publishing, Inc. Abstract: A procedure for tritiation of predominantly the cyclohexene moiety of abscisic acid was developed. Tritium was introduced by isotope exchange reaction with 100% tritiated water at 220°C in the presence of diisopropylethylamine. The yield of abscisic acid was 50%, and the specific activity was 30.5 Ci/mmol. The labeled product was tested. It was shown that tritiated abscisic acid synthesized by the proposed method did not differ from the unlabeled precursor and could be used for biological assays.

© 2018, Koninklijke Nederlandse Planteziektenkundige Vereniging. In the present study, we have monitored the process of systemic plant colonization by the plant pathogenic bacterium Pectobacterium atrosepticum (Pba) using RNA-Seq analysis in order to compare bacterial traits under in planta and in vitro conditions and to reveal potential players that participate in switching from stealth to brute force strategy of the pathogen. Two stages of tobacco plant colonization have been assayed: i) the initial one associated with visually symptomless spread of bacteria throughout the host body via primary xylem vessels where bacterial emboli were formed (stealth strategy), and ii) the advanced stage coupled with an extensive colonization of core parenchyma and manifestation of soft rot symptoms (brute force strategy). Plant-inducible genes in Pba and potential players switching the pathogen’s behavior were revealed. Genes from the cfa locus responsible for the production of coronafacic acid displayed the strongest induction in the asymptomatic zone relative to the symptomatic one and were shown experimentally to act as the true strategy “switchers” of Pba behavior in planta. Surprisingly, cfa genes appeared to be unnecessary for establishment of the asymptomatic stage of plant colonization but were required for the transition to soft-rot-associated symptomatic stage coupled with over-induction of jasmonate-mediated pathway in the plant.

© 2017 Gorshkov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the max

© 2016 German Botanical Society and The Royal Botanical Society of the NetherlandsRepresentatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant–Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific ‘multicellular’ structures – bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host–pathogen integration, since the formation of these structures requires the action of both partners.

© 2017, The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

© 2016 Institut Pasteur.Bacteria have high adaptive potential that ensures their survival during various environmental challenges. To adapt, bacteria activate a physiological program of stress response that makes them able to persist under adverse conditions. The present study sought to examine the ability of a particular bacterial species to induce a stress response in alternative scenarios. Cells of the phytopathogenic microorganism Pectobacterium atrosepticum were taken as a model. The cells were exposed to starvation in different physiological states (actively growing exponential phase and stationary phase cells), and the resulting starving cultures were monitored using CFU counting, quantitative PCR and electron microscopy. When exponential phase cells were subjected to starvation, the nucleoids of the cells became condensed and their DNA was detected by qPCR less effectively than that of cells growing in nutrient-rich medium, or stationary phase cells after starvation. Exponential phase cells subjected to starvation showed increased expression of genes encoding DNA binding histone-like proteins, whereas, in cultures inoculated by stationary phase cells, cell-wall-deficient forms that were inefficient at colony forming and that had a non-culturable phenotype were formed. The cell-wall-deficient forms displayed reduced expression of genes encoding synthases of cell wall components.

The adaptive reactions of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043 under starvation conditions were studied. The main emphasis was given to the peculiarities of stress responses depending on the bacterial population densities. When bacteria were subjected to starvation at high population densities (107-109CFUml-1), their adaptive reactions conformed to the conventional conception of bacterial adaptation related to autolysis of part of the population, specific modification of cell ultrastructure, activation of expression of stress responsive genes and acquiring cross protection against other stress factors. In contrast, at low initial population densities (103-105CFUml-1), as described in our recent work, the cell density increased due to multiple cell division despite the absence of exogenous growth substrate. Here we present data that demonstrate that such unconventional behavior is part of a stress response, which provides increased stress tolerance while retaining virulence. Cell morphology and gene expression in high- and low-cell-density starving Pba cultures were compared. Our investigation demonstrates the existence of alternative adaptive strategies enabling pathogenic bacteria to cope with a variety of stress factors, including starvation, especially necessary when residing outside of their host. © 2013 Institut Pasteur.

© 2017 Begell House, Inc. We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically ac tive stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim In the present study, we attempted to elucidate if the harmful phytopathogenic bacteria of Pectobacterium genus (P. atrosepticum) possess the enzymes for oxidation of phenolic compounds. Polyphenol oxidase (laccase) activity was revealed in P. atrosepticum cell lysates. Using bioinformatic analysis, an ORF encoding a putative copper-containing polyphenol oxidase of 241 amino acids with a predicted molecular mass of 25.9 kDa was found. This protein (named Pal1) shares significant level of identity with laccases of a new type described for several bacterial species. Cloning and expression of the pal1 gene and the analysis of corresponding recombinant protein confirmed that Pal1 possessed laccase activity. The recombinant Pal1 protein was characterized in terms of substrate specificity, kinetic parameters, pH and temperature optimum, sensitivity to inhibitors and metal content. Pal1 demonstrated alkali- and thermo-tolerance. The kinetic parameters K m and kcat for 2,6-dimethoxyphenol were 0.353 ± 0.062 mM and 98.79 ± 4.9 s −1 , respectively. The protein displayed high tolerance to sodium azide, sodium fluoride, NaCl, SDS and cinnamic acid. The transcript level of the pal1 gene in P. atrosepticum was shown to be induced by plant-derived phenolic compound (ferulic acid) and copper sulfate.

© 2017 Begell House, Inc. We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically ac tive stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

© The Author 2017. Published by Oxford University Press. All rights reserved. In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D "multicellular" structures-bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

© The Author 2017. Published by Oxford University Press. All rights reserved. In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D "multicellular" structures-bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim In the present study, we attempted to elucidate if the harmful phytopathogenic bacteria of Pectobacterium genus (P. atrosepticum) possess the enzymes for oxidation of phenolic compounds. Polyphenol oxidase (laccase) activity was revealed in P. atrosepticum cell lysates. Using bioinformatic analysis, an ORF encoding a putative copper-containing polyphenol oxidase of 241 amino acids with a predicted molecular mass of 25.9 kDa was found. This protein (named Pal1) shares significant level of identity with laccases of a new type described for several bacterial species. Cloning and expression of the pal1 gene and the analysis of corresponding recombinant protein confirmed that Pal1 possessed laccase activity. The recombinant Pal1 protein was characterized in terms of substrate specificity, kinetic parameters, pH and temperature optimum, sensitivity to inhibitors and metal content. Pal1 demonstrated alkali- and thermo-tolerance. The kinetic parameters K m and kcat for 2,6-dimethoxyphenol were 0.353 ± 0.062 mM and 98.79 ± 4.9 s −1 , respectively. The protein displayed high tolerance to sodium azide, sodium fluoride, NaCl, SDS and cinnamic acid. The transcript level of the pal1 gene in P. atrosepticum was shown to be induced by plant-derived phenolic compound (ferulic acid) and copper sulfate.

© 2017, The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

© 2017 Gorshkov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the max

© 2016 German Botanical Society and The Royal Botanical Society of the NetherlandsRepresentatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant–Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific ‘multicellular’ structures – bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host–pathogen integration, since the formation of these structures requires the action of both partners.

© 2016 Institut Pasteur.Bacteria have high adaptive potential that ensures their survival during various environmental challenges. To adapt, bacteria activate a physiological program of stress response that makes them able to persist under adverse conditions. The present study sought to examine the ability of a particular bacterial species to induce a stress response in alternative scenarios. Cells of the phytopathogenic microorganism Pectobacterium atrosepticum were taken as a model. The cells were exposed to starvation in different physiological states (actively growing exponential phase and stationary phase cells), and the resulting starving cultures were monitored using CFU counting, quantitative PCR and electron microscopy. When exponential phase cells were subjected to starvation, the nucleoids of the cells became condensed and their DNA was detected by qPCR less effectively than that of cells growing in nutrient-rich medium, or stationary phase cells after starvation. Exponential phase cells subjected to starvation showed increased expression of genes encoding DNA binding histone-like proteins, whereas, in cultures inoculated by stationary phase cells, cell-wall-deficient forms that were inefficient at colony forming and that had a non-culturable phenotype were formed. The cell-wall-deficient forms displayed reduced expression of genes encoding synthases of cell wall components.