Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum.

Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum. IMPORTANCE: About one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics. The special cell wall architecture of Corynebacteriales is critical for treatments because it is either a direct target or a barrier that the drug has to cross. Here, we present the analysis of LcpA and LcpB of the closely related Corynebacterium glutamicum, the first of which is an essential protein involved in cell wall biogenesis. Our work provides a comprehensive characterization of the impact of LCP proteins on cell wall biogenesis in this medically and biotechnologically important class of bacteria. Special focus is set on the two periplasmic LcpA domains and their contributions to physiological function.

Pyrones as bacterial signaling molecules.

Bacteria communicate via small diffusible molecules and thereby mediate group-coordinated behavior, a process referred to as quorum sensing. The prototypical quorum sensing system found in Gram-negative bacteria consists of a LuxI-type autoinducer synthase that produces N-acyl homoserine lactones (AHLs) as signals and a LuxR-type receptor that detects the AHLs to control expression of specific genes. However, many proteobacteria have proteins with homology to LuxR receptors yet lack any cognate LuxI-like AHL synthase. Here we show that in the insect pathogen Photorhabdus luminescens the orphan LuxR-type receptor PluR detects endogenously produced α-pyrones that serve as signaling molecules at low nanomolar concentrations. Additionally, the ketosynthase PpyS was identified as pyrone synthase. Reconstitution of the entire system containing PluR, the PluR-target operon we termed pcf and PpyS in Escherichia coli demonstrated that the cell-cell communication circuit is portable. Our research thus deorphanizes a signaling system and suggests that additional modes of bacterial communication may await discovery.

The lipid II flippase RodA determines morphology and growth in Corynebacterium glutamicum.

Lipid II flippases play an essential role in cell growth and the maintenance of cell shape in many rod-shaped bacteria. The putative lipid II flippase RodA is an integral membrane protein and member of the SEDS (shape, elongation, division and sporulation) protein family. In contrast to its homologues in Escherichia coli and Bacillus subtilis little is known about the role of RodA in actinobacteria. In this study, we describe the localization and function of RodA in Corynebacterium glutamicum, a rod-shaped, apically growing actinobacterium. RodA-GFP localizes exclusively at the cell poles. Surprisingly, time-lapse microscopy revealed that apical cell growth is sustained in a rodA deletion strain. However, growth rates and antibiotic susceptibility are altered. In the absence of RodA, it appears that apical growth is driven by lateral diffusion of lipid II that is likely flipped by the septal flippase, FtsW. Furthermore, we applied a previously described synthetic in vivo system in combination with FRET to identify an interaction between C. glutamicum RodA and the polar growth organizing protein DivIVA.

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope.

The mycomembrane (MM) is a mycolic acid layer covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. Biosynthesis of the MM is an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) and the new drug candidate, benzothiazinone 043 (BTZ) interfere with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of BTZ on the cell envelope. Our work shows that BTZ efficiently blocks the apical growth machinery, however effects in combinatorial treatment with ß-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. This layer was not detectable in the septa of BTZ or EMB treated cells. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG). While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis, slowing down growth effectively. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

Thalassospira sp. isolated from the oligotrophic eastern Mediterranean Sea exhibits chemotaxis toward inorganic phosphate during starvation.

The eastern Mediterranean Sea represents an ultraoligotrophic environment where soluble phosphate limits the growth of bacterioplankton. Correspondingly, genes coding for high-affinity phosphate uptake systems and for organophosphonate utilization are highly prevalent in the plankton metagenome. Chemotaxis toward inorganic phosphate constitutes an alternative strategy to cope with phosphate limitation, but so far has only been demonstrated for two bacterial pathogens and an archaeon, and not in any free-living planktonic bacterium. In the present study, bacteria affiliated with the genus Thalassospira were found to constitute a regular, low-abundance member of the bacterioplankton that can be detected throughout the water column of the eastern Mediterranean Sea. A representative (strain EM) was isolated in pure culture and exhibited a strong positive chemotaxis toward inorganic phosphate that was induced exclusively in phosphate-starved cultures. Phosphate-depleted cells were 2-fold larger than in exponentially growing cultures, and 43% of the cells retained their motility even during prolonged starvation over 10 days. In addition, Thalassospira sp. strain EM was chemotactically attracted by complex substrates (yeast extract and peptone), amino acids, and 2-aminoethylphosphonate but not by sugar monomers. Similarly to the isolate from the eastern Mediterranean, chemotaxis toward phosphate was observed in starved cultures of the other two available isolates of the genus, T. lucentensis DSM 14000T and T. profundimaris WP0211T. Although Thalassospira sp. represents only up to 1.2% of the total bacterioplankton community in the water column of the eastern Mediterranean Sea, its chemotactic behavior potentially leads to an acceleration of nutrient cycling and may also explain the persistence of marine copiotrophs in this extremely nutrient-limited environment.

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria.

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE: Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.

Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones.

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of alpha-glucosyl and D-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of D-alanine residues in the LTA backbones. Prior incubation of the LTAs with alpha-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of alpha-glucosyl-substituted, D-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption.

Sphingomonas kaistensis sp. nov., a novel alphaproteobacterium containing pufLM genes.

Three Gram-negative, non-motile, non-spore-forming short rods (strains PB56(T), PB180, PB229) were isolated from soil in South Korea. Cells were orange-red in colour. Strains PB180 and PB229 contained small amounts of bacteriochlorophyll a, which was not detected in strain PB56(T). However, all three isolates contained the genes for the photosynthetic type II reaction centre, pufLM. They contained Q-10 as the dominant quinone and C(18 : 1) as the dominant fatty acid. The highest 16S rRNA gene sequence similarities were found to Sphingomonas oligophenolica JCM 12082(T) (95.8 %), Sphingomonas koreensis KCTC 2882(T) (95.1 %), Sphingomonas mali IFO 15500(T) (95.1 %), Sphingomonas faeni DSM 14747(T) (94.8 %), Sphingomonas pruni IFO 15498(T) (94.7 %) and Sphingomonas aquatilis KCTC 2881(T) (94.6 %), as well as to Sphingosinicella microcystinivorans Y2(T) and Sphingosinicella xenopeptidilytica 3-2W4(T) (95.0-95.2 %). Phylogenetic analyses supported the assignment of strains PB56(T), PB180, PB229 to the genus Sphingomonas. The novel isolates differ from all established species of the genus Sphingomonas by their higher G+C content and the absence of straight-chain 2-hydroxy fatty acids. Based on the phylogenetic distances from species with validly published names and their phenotypic properties, the strains constitute a separate species, for which the name Sphingomonas kaistensis sp. nov. is proposed. The type strain is PB56(T) (=KCTC 12334(T)=DSM 16846(T)).

Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments.

Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic-rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon-lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter, formerly classified as Agrobacterium tumefaciens. To prove and quantify the in situ abundance of R. radiobacter, a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25-40 000 cells per gram of sediment were detected, corresponding to 0.001-5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment.

Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community.

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.

Phototrophic consortia: model systems for symbiotic interrelations between prokaryotes.

Most symbiotic prokaryotes known to date have been found in association with eukaryotes, whereas only few (3.5%) bacteria or archaea are known that have established interactions with other prokaryotes. As revealed by direct microscopic investigations, however, multiple morphotypes of highly structured associations of different prokaryotes exist in nature. These so-called consortia appear to represent the most developed type of bacterial interaction. Phototrophic consortia are associations of green sulfur bacteria that surround a central chemotrophic bacterium. In some natural environments, almost all cells of green sulfur bacteria occur in the associated state, i.e. as epibionts of phototrophic consortia. In contrast to earlier speculations, the central bacterium belongs to the beta-Proteobacteria. Within the consortia, the green sulfur bacterial epibionts grow photolithoautotrophically, utilizing exogenous sulfide as photosynthetic electron donor. The entire consortium does not appear to be independent of organic carbon compounds, since it exhibits chemotaxis towards 2-oxoglutarate and, as demonstrated by microautoradiography, can also incorporate this compound. Intact consortia exhibit a scotophobic response in which the bacteriochlorophylls of the epibionts function as light sensors, whereas the chemotrophic central bacterium confers motility upon the association. Hence, a signal exchange must occur between the different bacteria. Based on their 16S rRNA gene sequences, the epibionts represent distinct phylotypes that are often only distantly related to known species of green sulfur bacteria. Since phototrophic consortia have recently become available in enrichment cultures, they can now serve as suitable model systems for the investigation of the molecular mechanisms of cell-cell recognition and signal exchange, and for studies of the coevolution of nonrelated prokaryotes.

Characterization of lipoteichoic acids as Lactobacillus delbrueckii phage receptor components.

Lipoteichoic acids (LTAs) were purified from Lactobacillus delbrueckii subsp. lactis ATCC 15808 and its LL-H adsorption-resistant mutant, Ads-5, by hydrophobic interaction chromatography. L. delbrueckii phages (LL-H, the LL-H host range mutant, and JCL1032) were inactivated by these poly(glycerophosphate) type of LTAs in vitro in accordance to their adsorption to intact ATCC 15808 and Ads-5 cells.