Validation of IRS PCR, a molecular typing method, for the study of the diversity and population dynamics of Legionella in industrial cooling circuits.

Legionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed-field gel electrophoresis (PFGE) and sequence-based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

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.

Intragenic Suppression at the b2 Locus in ASCOBOLUS IMMERSUS. II. Characteristics of the Mutations in Groups A and E.

The present report is a study of 60 intragenic suppressors located in the A or E group of the b2 spore-color locus of Ascobolus immersus. The frameshift nature of the suppressors was shown by 19 combinations of plus and minus suppressor mutations. The location of the mutation sites on the genetic map of group A was defined. The conversion pattern of the frameshift suppressors is discussed according to their plus or minus phase shift and their mutagenic origin.

Intragenic suppression at the b2 locus in Ascobolus immersus. I. Identification of three distinct groups of suppression.

A reversion study of EMS- or ICR170-induced ascospore color mutants in Ascobolus immersus is reported. Twenty-three new intragenic suppressors were isolated within the b2 locus. These are localized within three distinct groups on a genetic fine-structure map containing 21 identifiable sites. The pattern of reversion is discussed according to the known specificity of EMS and ICR170 in Ascobolus immersus.

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions.

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.

Improved electro-transformation of highly DNA-restrictive corynebacteria with DNA extracted from starved Escherichia coli.

Differences of up to 33 000-fold in electro-transformability of highly DNA restrictive corynebacteria are observed in the DNA of a shuttle plasmid extracted from Escherichia coli hosts propagated in different nutritional conditions. Growth of the host in minimal medium increases plasmid transformability, whereas growth on rich media decreases it. In the E. coli DH5 alpha host, the starvation-dependent increase DNA transformability is reverted by supplementing with methionine, an obligate 5-adenosyl-methionine (SAM) precursor. This suggests that an E. coli nutritionally modulated SAM-dependent DNA-methyltransferase may be involved in this phenomenon.

Positively regulated expression of the Escherichia coli araBAD promoter in Corynebacterium glutamicum.

In Corynebacterium glutamicum the promoter of the araBAD Escherichia coli gene is positively regulated by both arabinose and the araC gene product, as it is the case in the natural host. If the L-arabinose inducer and an active araC gene are present, significant amounts of araBAD promoter expression take place in the absence of the E. coli CRP protein. These results show that the C. glutamicum RNA polymerase is activated by the E. coli positive regulator of transcription AraC.

S-layer protein transport across the cell wall of Corynebacterium glutamicum: in vivo kinetics and energy requirements.

Corynebacteria are Gram-positive bacteria with a very peculiar cell envelope structure as it is constituted of an inner membrane and an outer membrane-like structure. Protein secretion in Corynebacterium glutamicum was studied in vivo, using the S-layer protein PS2 as a model. We show that different variants of PS2 protein are exported through the whole cell envelope with a half-life ranging between 2 and 4 min, by a two-step mechanism. The first step, which is over after about 1.5 min, is ATP- and proton motive force-dependent and may correspond to translocation across the inner membrane via the 'Sec' machinery. The second step, across the cell wall and the outer mycolate layer, is rapid but independent of energy sources. This very efficient secretion process across the mycolate layer raises the question of the existence in this layer of a specific machinery.

Interspecies electro-transformation in Corynebacteria.

Plasmid DNA was efficiently electro-transformed into intact cells of nine Corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum and Corynebacterium melassecola. Relationships were explored between transformation efficiency and parameters such as electric field strength and pulse length, DNA concentration, physiological state and concentration of the cells. In optimal conditions, more than 10(7) transformants per microgram of DNA could be obtained. Electro-transformation with plasmid DNA isolated from different sources indicates that DNA modification may play a role in transformation efficiency.

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.

A system for the detection of chromosomal rearrangements using Sordaria macrospora.

A system is described for the detection and diagnosis of induced chromosomal rearrangement using Sordaria macrospora. The system uses the property of the rearrangement to produce defective white ascospores as meiotic progeny from heterozygous crosses. Two reconstruction experiments have shown that this system is able to give reliable quantitative measures of rearrangement frequencies. Evidence for a photoreactivation process was obtained, suggesting that pyrimidine dimers may well be an important lesion in UV-induced chromosomal rearrangement. No evidence of induction of chromosomal rearrangement was obtained in experiments with the powerful chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine.

An ATP-based method for monitoring the microbiological drinking water quality in a distribution network.

The titration of adenosine triphosphate (ATP) by bioluminescence permits rapid evaluation of the quantity of viable micro-organisms present in a water sample. During two sampling campaigns, Société Anonyme de Gestion des Eaux de Paris (SAGEP) tested a new extraction and titration system of bacterial ATP in the Paris drinking water distribution network. As far as the entire set of results of analyses of water in the network is concerned there is a linear relationship between log [ATP] and log(HPC-R2A/ml). Furthermore, as regards the drinking water originating from treatment of surface waters, some of the results obtained indicate a slight change as regards the Paris network in the microbiological quality. This is certainly linked to the distance travelled from the production location as well as to a reservoir effect observed on a site. Conversely, no change is apparent with regard to waters of underground origin. Lastly, despite changes in temperature and chlorine residual, no significant influence has been observed, essentially because of the very low density of culturable bacteria.

Corrected gene assignments of Escherichia coli pro- mutations.

We have reevaluated the gene assignments of the proline mutant alleles of some known Pro- Escherichia coli strains. Of nine proline auxotrophs included in the study, five presented phenotypes inconsistent with their previously assigned genotypes. We discuss the possible sources and the consequences of these assignment errors.

Electrotransformation of highly DNA-restrictive corynebacteria with synthetic DNA.

Highly DNA-restrictive Corynebacteria can be transformed with DNA made in vitro by PCR amplification of a sequence that contains the replication origin of pBL1, a plasmid common to many Corynebacteria. In all strains examined, the transformation efficiencies of PCR-synthetized DNA equal or improve the performances of heterologous DNA extracted from wild-type and dam(-)-dcm-strains of Escherichia coli. The transformation efficiencies obtained with PCR-made DNA may be high enough to permit its general application to experiments of gene integration.

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 al., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290 bp long, carries 32 bp imperfect inverted repeats and generates a 3 bp 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 IS1206 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.

Multicopy suppression by asd gene and osmotic stress-dependent complementation by heterologous proA in proA mutants.

Auxotrophic proA mutants of Escherichia coli were complemented by two different classes of Corynebacterium glutamicum genes. One of these was the asd gene. The E. coli asd gene also complements the same proA alleles. Complementation of proA by the asd+ gene requires a high asd dosage and the proB and the proC gene products. The reciprocal complementation pattern (asd by the proA+ gene) was not observed. This complementation appears to be due to multicopy suppression by a proline biosynthetic gene whose product was expected to play a negligible role in this pathway. The other class of complementing clones carries the C. glutamicum proA gene. Complementation of E. coli proA mutants by the C. glutamicum proA+ gene was optimal at high osmolarity.

Indirect intergenic suppression of a radiosensitive mutant of Sordaria macrospora defective in sister-chromatid cohesiveness.

Six ultra violet (UV) mutageneses were performed on the spo76 UV-sensitive mutant of Sordaria macrospora. Spo76 shows an early centromere cleavage associated with an arrest at the first meiotic division and therefore does not form ascospores. Moreover, it exhibits altered pairing structure (synaptonemal complex), revealing a defect in the sister-chromatid cohesiveness. From 37 revertants which partially restored sporulation, 34 extragenic suppressors of spo76 were isolated. All suppressors are altered in chromosomal pairing but, unlike spo76, show a wild type centromere cleavage. The 34 suppressors were assigned to six different genes and mapped. Only one of the suppressor genes is involved in repair functions.