Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions.

DNA-SIP (stable isotope probing) was conducted on anaerobic municipal solid waste samples incubated with (13)C-cellulose, (13)C-glucose and (13)C-acetate under mesophilic conditions. A total of 567 full-length bacterial and 448 1100-bp-length archaeal 16S rRNA gene sequences were analysed. In the clone libraries derived from 'heavy' DNA fractions, the most abundant sequences were affiliated with the phyla Firmicutes, Bacteroidetes, the gamma-subclass of Proteobacteria and methanogenic orders Methanomicrobiales and Methanosarcinales. Sequences related to the genus Acetivibrio (phylum Firmicutes) were recovered only in the 'heavy' DNA fraction derived from the (13)C-cellulose incubation. An oligonucleotide probe (UCL284) targeting specifically Acetivibrio was designed and used for fluorescent in situ hybridization (FISH) experiments. Interestingly, hybridization of the probe was detected in microorganisms aggregated around cellulose fibres, strengthening the conclusion that these microorganisms were major cellulose degraders. Sequences related to genus Clostridium (phylum Firmicutes) and to the family Porphyromonadaceae (phylum Bacteroidetes) were retrieved in large numbers from the 'heavy' DNA library of (13)C-Glucose incubation, suggesting their involvement in saccharide fermentation. Design and hybridization of specific FISH-probes confirmed the abundant representation of Clostridium (CLO401, CLO1248) and Porphyromonadaceae (BAC1040), which were mostly observed in the planktonic phase. Surprisingly, in the (13)C-acetate experiment, the 'heavy' DNA archaeal library was dominated by sequences related to the strictly hydrogenotrophic methanogenic genus Methanoculleus. One single operational taxonomic unit containing 70 sequences, affiliated to the gamma-subclass of Proteobacteria, was retrieved in the corresponding bacterial library. FISH observations with a newly designed specific probe (UGA64) confirmed the dominance of this bacterial group. Our results show that combination of DNA-SIP and FISH applied with a series of functionally connected substrates can shed light on the networks of uncultured microbes catalysing the methanization of the most abundant chemical renewable energy source on Earth.

Simultaneous analysis of microbial identity and function using NanoSIMS.

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.

New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum.

Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope. We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C. The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt). In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C. glutamicum and three cmyt genes in C. diphtheriae. In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis. In C. glutamicum CGL2005 cmytE is a pseudogene. The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains. Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain. For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene. Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.

Promoters of Corynebacterium glutamicum.

Regulation of gene expression in Corynebacterium glutamicum represents an important issue since this Gram-positive bacterium is a notable industrial amino acid producer. Transcription initiation, beginning by binding of RNA polymerase to the promoter DNA sequence, is one of the main points at which bacterial gene expression is regulated. More than 50 transcriptional promoters have so far been experimentally localized in C. glutamicum. Most of them are assumed to be promoters of vegetative genes recognized by the main sigma factor. Although transcription initiation rate defined by many of these promoters may be affected by transcription factors, which activate or repress their function, the promoter regions share common sequence features, which may be generalized in a consensus sequence. In the consensus C. glutamicum promoter, the prominent feature is a conserved extended -10 region tgngnTA(c/t)aaTgg, while the -35 region is much less conserved. Some commonly utilized heterologous promoters were shown to drive strong gene expression in C. glutamicum. Conversely, some C. glutamicum promoters were found to function in Escherichia coli and in other bacteria. These observations suggest that C. glutamicum promoters functionally conform with the common bacterial promoter scheme, although they differ in some sequence structures.

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.

Susceptibility of Legionella strains to the chlorinated biocide, monochloramine.

Members of the Legionella genus find suitable conditions for their growth and survival in nuclear power plant cooling circuits. To limit the proliferation of Legionella pathogenic bacteria in nuclear power plant cooling circuits, and ensure that levels remain below regulatory thresholds, monochloramine treatment can be used. Although the treatment is highly effective, i.e. it reduces Legionella numbers by over 99%, Legionella bacteria can still be detected at low concentrations and rapid re-colonisation of circuits can occur after the treatment has ceased. The aim of this study was to develop an in vitro methodology for determining the intrinsic susceptibility of L. pneumophila strains, collected from various nuclear power plant cooling circuits subjected to different treatment conditions. The methodology was developed by using an original approach based on response surface methodology (RSM) combined with a multifactorial experimental design. The susceptibility was evaluated by the Ct factor. The susceptibility of environmental strains varies widely and is, for some strains, greater than that of known tolerant species; however, strain susceptibility was not related to treatment conditions. Selection pressure induced by monochloramine use did not result in the selection of more tolerant Legionella strains and did not explain the detection of Legionella during treatment or the rapid re-colonisation of cooling circuits after disinfection has ceased.

The impact of monochloramine on the diversity and dynamics of Legionella pneumophila subpopulations in a nuclear power plant cooling circuit.

Members of the pathogenic Legionella genus encounter suitable growth conditions in nuclear power plant cooling circuits. To limit its proliferation and ensure that levels remain below regulatory thresholds, chemical treatment with monochloramine can be used in continuous or sequential conditions. The aim of this study was to determine the impact of monochloramine on L. pneumophila subpopulations in the cooling circuits of a nuclear power plant. The chosen procedure involved monitoring the diversity and dynamics of L. pneumophila subpopulations every month over the course of a year in a nuclear power plant cooling circuit, which was treated for 2 months during the period under study. This study confirmed the effectiveness of monochloramine to limit L. pneumophila concentrations in cooling circuits. The culturable L. pneumophila community was strongly affected by the injection of monochloramine. Several subpopulations persisted during treatment at low concentrations (below the detection limit of standard methods), suggesting that the susceptibility of L. pneumophila is strain dependent. Although the composition of the subpopulations was not similar, the resilience of the community structure was observed. Indeed, the community eventually returned to its initial structure and presented a similar pattern of richness, diversity and uniformity to that seen before treatment.

A simple system for biofilm potential monitoring in drinking water.

SAGEP-EAU DE PARIS produces drinking water for the city of Paris (France). In order to supply a high quality water, one of the main SAGEP's concerns is to monitor the Biofilm Formation Potentials of the produced drinking waters. Biofilm incubators were installed at the outlet of three Water Treatment Plants (WTP). These incubators allowed biofilm formation and quantification in terms of Fixed Total Organic Carbon (FTOC), fixed culturable bacteria (HPC-R2A) and fixed total bacteria. During this study, quantitative differences appeared between the biofilms formed at the outlet of the three WTPs, leading to different classifications of the Biofilm Formation Potentials of the three produced waters, depending on the used parameter for biofilms quantification. This observation underlined the necessity of a multi-parametric approach for the study of biofilms. More generally, our results validated the use of these sturdy stainless steel incubators, highly adapted to industrial field conditions, for the monitoring of Biofilm Formation Potentials in drinking water networks.

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.

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.

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.