Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

[CORYNEBACTERIUM: FEATURES OF THE STRUCTURE OF THE BACTERIAL CELL].

In a review of the features of the bacterial cells are Corynebacterium structure: characterized by an upper layer, highly organized cell wall, cytoplasmic membrane, cytoplasm, nucleoid. Described in detail the structure of the upper layer containing pili (fimbriae), microcapsule surface proteins - PS-2, DIP1281, 67-72r protein (hemagglutinin), porins, sialidase (neuraminidase). These components are the ability to initiate a serial of Corynebacterium work with the host cell, followed by colonization. It submitted a detailed description .of the structure and functions of cell wall structures - cord factor, which is a second barrier permeability; arabinogalactan, peptidoglycan, lipomannan and lipoarabinomannan. The structure and function of the cytoplasmic membrane as the main diffusion barrier cell cytoplasm and the genome of Corynebacterium. Presented differ- ent molecular genetic methods for the identification and differentiation of closely related species of Corynebacterium.

Colonization of human epithelial cell lines by Corynebacterium ulcerans from human and animal sources.

Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the colonization of the human host are lacking up to now. In this study, adhesion of two C. ulcerans isolates to human epithelial cells, invasion of host cells and the function of two putative virulence factors with respect to these processes were investigated. C. ulcerans strains BR-AD22 and 809 were able to adhere to Detroit562 and HeLa cells, and invade these epithelial cell lines with a rate comparable to other pathogens as shown by scanning electron microscopy, fluorescence microscopy and replication assays. Infection led to detrimental effects on the cells as deduced from measurements of transepithelial resistance. Mutant strains of putative virulence factors phospholipase D and DIP0733 homologue CULC22_00609 generated in this study showed no influence on colonization under the experimental conditions tested. The data presented here indicate a high infectious potential of this emerging pathogen.

Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak.

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

[THE COMPARATIVE ANALYSIS OF TECHNIQUES OF IDENTIFICATION OF CORYNEBACTERIUM NON DIPHTHERIAE].

The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

[Dormant state and phenotypic variability of Staphylococcus aureus and Corynebacterium pseudodiphtheriticum].

Ability to produce dormant forms (DF) was demonstrated for non-spore-forming bacteria Staphylococcus aureus (a nonpathogenic strain) and Corynebacterium pseudodiphtheriticum (an organism of the normal oropharyngeal flora). The salient features of the sthaphylococcal and corynebacterial DF were (1) prolonged preservation of viability; (2) resistance to damaging factors (heat treatment); and (3) specific morplology and ultrastructure. The optimal conditions for DF formation were (1) transfer of the stationary-phase cultures into saline solution with CaCl2 (10-300 mM) (for S. aureus); (2) growth in SR1 synthetic medium with fivefold nitrogen limitation (for C. pseudodiphtheriticum); and (3) incubation with (1-5) x 10(-4) M) of C12-AHB, an alkylhydroxybenzene akin to microbial anabiosis autoinducers. Increase of C12-AHB concentration to 7 x 10(-4) -2 x 10(-3) M resulted in "mummification" with irreversible loss of viability without autolytic processes. Germination of the dormant forms was followed by increased phenotypic variability, as seen from (1) diversity of colony types and (2) emergence of antibiotic-resistant clones on selective media. The share of kanamycin-resistant S. aureus variants was most numerous 0.002-0.01% in 4-month DF suspensions in saline with CaCl2. In the C. pseudodiphtheriticum DF produced under the effect of C12-AHB, the share of kanamycin-resistant variants was also found to increase. These data point to association between emergence of antibiotic-resistant variants and their persistence in dormant state mediated by starvation stress and regulated by AHB.

Physiological and biochemical consequences of host-plasmid interaction--a case study with Corynebacterium renale, a multiple cryptic plasmid containing strain.

Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, µ, and respiratory rate, r. Both µ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in µ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

El genoma del corynebacterium urealyticum ha sido completamente secuenciado: un progreso clave para conocer la uropatogenicidad y "estilo de vida" de este microorganismo / The Corynebacterium urealyticum genome has been completely sequenced: a key advance to know the uropathogenicity and «life style» of this microorganism

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Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.

The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergent-treated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Guérin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.

Overproduction of NAD+ and 5'-inosine monophosphate in the presence of 10 microM Mn2+ by a mutant of Corynebacterium ammoniagenes with thermosensitive nucleotide reduction (nrd(ts)) after temperature shift.

Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction ( nrd(ts)). The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures. No overproduction of NAD(+) was detected in the control maintained at 27 degrees C whereas NAD(+) was accumulated extracellularily by strain CH31 at 37 degrees C and at 40 degrees C. As a result of the temperature shift, division-inhibited cells displayed only limited elongation. This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria. Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD(+)-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication. In contrast to the low extracellular accumulation of NAD(+), at the non-permissive temperature of 35 degrees C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells. The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 microM Mn(2+). Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5'-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.

Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum.

Mycoloyltransferases (Myts) play an essential role in the biogenesis of the cell envelope of members of the Corynebacterineae, a group of bacteria that includes the mycobacteria and corynebacteria. While the existence of several functional myt genes has been demonstrated in both mycobacteria and corynebacteria (cmyt), the disruption of any of these genes has at best generated cell-wall-defective but always viable strains. To investigate the importance of Myts on the physiology of members of the Corynebacterineae, a double mutant of Corynebacterium glutamicum was constructed by deleting cmytA and cmytB, and the consequences of the deletion on the viability of the mutant, the transfer of corynomycoloyl residues onto its cell-wall arabinogalactan and trehalose derivatives, and on its cell envelope ultrastructure were determined. The double mutant strain failed to grow at 34 degrees C and exhibited a growth defect and formed segmentation-defective cells at 30 degrees C. Biochemical analyses showed that the double mutant elaborated 60 % less cell-wall-bound corynomycolates and produced less crystalline surface layer proteins associated with the cell surface than the parent and cmytA-inactivated mutant strains. Freeze-fracture electron microscopy showed that the DeltacmytA DeltacmytB double mutant, unlike the wild-type and cmytA-inactivated single mutant strains, frequently exhibited an additional fracture plane that propagated within the plasma membrane and rarely exposed the S-layer protein. Ultra-thin sectioning of the double mutant cells showed that they were totally devoid of the outermost layer. Complementation of the double mutant with the wild-type cmytA or cmytB gene restored completely or partially this phenotype. The data indicate that Myts are important for the physiology of C. glutamicum and reinforce the concept that these enzymes would represent good targets for the discovery of new drugs against the pathogenic members of the Corynebacterineae.

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications.

Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall. These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria. In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan-peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure. In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier. An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum. These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents. All these data address new aspects regarding cell wall permeability towards macromolecules and amino acids but also open new opportunities for biotechnology applications.

Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid.

Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.

Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain.

Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope.

Bacterial surface layers (S-layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments. Atomic force microscopy (AFM) was used to asses the S-layer of Coryne-bacterium glutamicum formed of PS2 proteins that assemble into hexameric complexes within a hexagonal lattice. Native and trypsin-treated S-layers were studied. Using the AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers were separated. All surfaces of native and protease-digested S-layers were imaged at better than 1 nm lateral resolution. Difference maps of the topographies of native and proteolysed samples revealed the location of the cleaved C-terminal fragment and the sidedness of the S-layer. Because the corrugation depths determined from images of both sides span the total thickness of the S-layer, a three-dimensional reconstruction of the S-layer could be calculated. Lattice defects visualized at 1 nm resolution revealed the molecular boundaries of PS2 proteins. The combination of AFM imaging and single molecule force spectroscopy allowed the mechanical properties of the Corynebacterium glutamicum S-layer to be examined. The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.

Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.

With the recent success of the heterologous expression of mycobacterial antigens in corynebacteria, in addition to the importance of these bacteria in biotechnology and medicine, a better understanding of the structure of their cell envelopes was needed. A combination of molecular compositional analysis, ultrastructural appearance and freeze-etch electron microscopy study was used to arrive at a chemical model, unique to corynebacteria but consistent with their phylogenetic relatedness to mycobacteria and other members of the distinctive suprageneric actinomycete taxon. Transmission electron microscopy and chemical analyses showed that the cell envelopes of the representative strains of corynebacteria examined consisted of (i) an outer layer composed of polysaccharides (primarily a high-molecular-mass glucan and arabinomannans), proteins, which include the mycoloyltransferase PS1, and lipids; (ii) a cell wall glycan core of peptidoglycan-arabinogalactan which may contain other sugar residues and was usually esterified by corynomycolic acids; and (iii) a typical plasma membrane bilayer. Freeze-etch electron microscopy showed that most corynomycolate-containing strains exhibited a main fracture plane in their cell wall and contained low-molecular-mass porins, while the fracture occurred within the plasma membrane of strains devoid of both corynomycolate and pore-forming proteins. Importantly, in most strains, the amount of cell wall-linked corynomycolates was not sufficient to cover the bacterial surface; interestingly, the occurrence of a cell wall fracture plane correlated with the amount of non-covalently bound lipids of the strains. Furthermore, these lipids were shown to spontaneously form liposomes, indicating that they may participate in a bilayer structure. Altogether, the data suggested that the cell wall permeability barrier in corynebacteria involved both covalently linked corynomycolates and non-covalently bound lipids of their cell envelopes.

S-layer protein production by Corynebacterium strains is dependent on the carbon source.

Three strains of Corynebacterium producing various amounts of PS2 S-layer protein were studied. For all strains, more PS2 was produced if the bacteria were grown in minimal medium supplemented with lactate than if they were grown in minimal medium supplemented with glucose. The consumption of substrate and PS2 production was studied in cultures with mixed carbon sources. It was found that the inhibitory effect of glucose consumption was stronger than the stimulatory effect of lactate in one strain, but not in the other two strains. The regulation of gene expression involved in S-layer formation may involve metabolic pathways, which probably differ between strains. S-layer organization was also studied by freeze-fracture electron microscopy. It was found that low levels of PS2 production correlated with the partial covering of the cell surface by a crystalline array. Finally, it was found that PS2 production was mainly regulated by changes in gene expression and that secretion was probably not a limiting step in PS2 accumulation.

Different modes of diaminopimelate synthesis and their role in cell wall integrity: a study with Corynebacterium glutamicum.

In eubacteria, there are three slightly different pathways for the synthesis of m-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is Corynebacterium glutamicum, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone dapD and prove by enzyme experiments that this gene encodes the succinylase (M(r) = 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation, dapD, as well as dapE encoding the desuccinylase, was inactivated, thereby forcing C. glutamicum to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.