Gérard Leblon

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.

The S‐layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C‐terminal hydrophobic domain

PS2 is the S‐layer protein of Corynebacterium glutamicum. The S‐layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S‐layer, which remains organized. Because we show that trypsin cleaves the C‐terminal part of the protein, we conclude that this domain is involved in the association of the S‐layer to the cell but is not essential in the interaction between individual PS2 proteins within the S‐layer. A modified form of PS2, deleted of its C‐terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S‐layer and is mainly released into the medium. We suggest that PS2 is anchored via its C‐terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

Summary— The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze‐fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimension as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced the ordered fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

Relevance of the INT test response as an indicator of ETS activity in monitoring heterotrophic aerobic bacterial populations in activated sludges

Abstract The activity of electron transport systems (ETS) assayed with 2-para (iodo-phenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) has been considered to be a specific indicator of the respiration of aerobic microorganisms and an appropriate way to monitor such populations in activated sludges. We have attempted to define the conditions for measuring ETS activity, as well as its relevance in monitoring these systems. We measured INT response and quantified microorganism populations in activated sludges under extreme conditions, such as oxygen starvation and deflocculation. We also measured the INT response in each of the following microbial populations: aerobic bacteria, anaerobic heterotrophic bacteria, protozoans, and metazoans, as well as in fragments of disrupted bacteria. The results reveal that strictly anaerobic bacteria in activated sludge also have a positive response to the INT test, which increases when flocs are dissociated or bacteria are disrupted. The INT reagent seems to have limited access to electron transport chains. The INT test in activated sludges thus does not appear to be suitable for monitoring the metabolism of aerobic heterotrophic bacteria, their principal population. The INT test therefore seems to be inappropriate for detecting a shift towards anaerobiosis.

Identification of IS1206, a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis.

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3‐related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.

Reviviscence of aerobic chemoheterotrophic bacteria in an activated sludge pilot plant after a prolonged absence of oxygen

The effect of the absence of oxygen (anoxia followed by prolonged anaerobiosis) on the dynamics and reviviscence of populations of aerobic chemoheterotrophic bacteria has been studied in the activated sludge of a pilot plant fed with a synthetic substrate (Viandox). The majority of the bacteria were Zoogloea, Alcaligenes and Pseudomonas, accounting for 65% to 85% of the cultivable species. Most of these bacteria are reviviscent after eight days of anaerobiosis, their relative proportion remaining practically unchanged. The protozoa and metazoa usually present in the sludge disappear over the first three days of absence of oxygen, to be replaced by species of the class Diplomonadida, an anaerobic phylum. In a pure culture, the Zoogloea and Alcaligenes strains from the pilot sludge remain reviviscent after 8 days without oxygen, but much less than in the pilot plant.

Role of the protonmotive force and of the state of the lipids in the in vivo protein secretion in Corynebacterium glutamicum, a Gram-positive bacterium

PS1 is a protein translocated across the cytoplasmic membrane of Corynebacterium glutamicum, a Gram-positive bacterium. Western blots of whole cell extracts showed the presence of two bands associated with the mature and the precursor forms. Addition of chloramphenicol led to the disappearance of the precursor form while dissipation of the protonmotive force (delta microH) prior to the addition of chloramphenicol prevented the maturation of the precursor. Dissipation of delta microH prior to a pulse chase experiment resulted in a complete block on translocation; regeneration of delta microH allowed the translocation of PS1 synthesized in its absence. On the other hand, dissipation of delta microH immediately after a pulse period had little effect on PS1 secretion. Lowering the temperature to 10 degrees C at the end of the pulse period completely inhibited secretion. The efficiency of secretion as a function of increasing temperature followed closely the order-to-disorder transition of the membrane lipids as detected by fluorescence anisotropy of diphenylhexatriene. Taken together, the results show that delta microH and the state of the lipids affect different steps of PS1 secretion.

The use of high halide-ion concentrations and automated phasing procedures for the structural analysis of BclA, the major component of the exosporium of Bacillus anthracis spores.

The structure determination of the recombinant form of BclA, the major protein component of Bacillus anthracis exosporium, involved soaking in a high concentration of potassium iodide as the means of obtaining a good-quality heavy-atom derivative. The data to 2 angstroms resolution collected on a laboratory source were of sufficient quality to allow successful phasing and chain tracing by automated methods.