Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.     

Identification and characterization of a heme periplasmic-binding protein in <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis>

Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Antibiotic resistance of potential probiotic bacteria of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.     

A study of the nutritional requirements of a selectedHaemophilus ducreyi strain by impedance and conventional methods

The growth and nutritional requirements ofHaemophilus ducreyi (CIP 542^T) were determined in liquid media by impedance technology. We found thatH. ducreyi grew in a liquid mixture of Proteose Peptone No. 3, hemin,l-serine, glucose,l-glutamine, and inorganic salts. The peptone in the medium was essential forH. ducreyi growth, and the growth factor (or factors) in the peptone was stable to autoclaving (15 min, 121°C) and could not be extracted with organic solvents (21 chloroform/methanol) from either acidic (1N CH₃COOH), neutral (pH 7.1), or basic (1N NaOH) solutions. Hemin was also an essential component for the growth ofH. ducreyi, but a much lower concentration of hemin (5.0 g/ml) was required than has been previously reported in the literature (25 g/ml). Using impedance technology to measureH. ducreyi doubling times, we showed that bovine albumin enhancesH. ducreyi growth.     

Facile construction of mutations in Haemophilus ducreyi using lacZ as a counter-selectable marker

Haemophilus ducreyi is a Gram-negative bacterium which is the causative agent of chancroid, an ulcerative sexually transmitted disease. In order to understand the pathogenesis of H. ducreyi disease, studies designed to identify potential virulence determinants and construct mutants deficient in the elaboration of these determinants have been undertaken in several laboratories. At the present time, construction of isogenic mutants is accomplished by electroporation of linearized DNA containing insertionally inactivated H. ducreyi genes followed by selection for the resistance marker encoded on the inactivated gene. In our experience, certain mutants are difficult to construct using this procedure. In the construction of strains containing lacZ as a reporter gene, we observed that the growth of lacZ expressing H. ducreyi was inhibited in the presence of X-gal. We have exploited this observation to develop a new strategy for the construction of isogenic H. ducreyi mutants.     

Allelic Polymorphism of the tet(M) Determinant in Mycoplasma hominis and Ureaplasma urealyticum Clinical Isolates Resistant to Tetracyclines

Of the 130 clinical isolates of Mycoplasma hominisfrom patients with nonspecific inflammatory diseases of the urogenital tract (UGT), approximately 10% contained the tet(M) gene after the course of treatment with tetracyclines. This gene was found in nine (25%) of the 36 Ureaplasma urealyticum clinical isolates. The nucleotide sequence of 13 tet(M) genes in Tc^R clinical isolates ofM. hominis and five genes in U. urealyticum Tc^R clinical isolates was determined. A comparison of nucleotide sequences of eight tetM genes of different origin and tet(M) genes ofGardnerella vaginalis and M. hominis and U. urealyticumclinical isolates showed that the mosaic structure of thetet(M) gene is completely identical in 11 of 13 M. hominis Tc^Risolates but belongs to an unidentified allele different from those described earlier. Another new allelic variant oftet(M) was found in two isolates. In three of five Tc^R clinical isolates of U. urealyticum, a tet(M) gene, whose mosaic structure was identical to that of tet(M) reported previously for ureaplasmas, and also two new allelic variants, which have not been described so far, were found.     

A role for <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis> Cu,ZnSOD in resistance to heme toxicity

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.     

Effects of Lac Operon Activation,Deletion of the Yhha Gene,and the Removal of Oxygen on the Ultra-Weak Photon Emission of Escherichia coli

We observed a relation between gene activity and ultra-weak photon emission (UPE). By comparing the UPEs of E. coli with the LacI gene present and deleted we found that more gene activity produced higher UPE. This relation was further confirmed by studying the UPE of the E. coli with and without the Yhha gene. We interpreted that a higher aminoacyl t-RNA synthetase activity, which used ATP from the respiratory chain, could increase the emission. Satisfying the increased need of ATP by the E. coli through an increase of respiratory chain activity, which has reactive oxygen species (ROS) as a byproduct, results in a higher rate of photon emission. To ensure that oxygen is at the origin of this emission, we replaced the air by pure nitrogen. After 30?min, it was observed that the emission levels equaled the emission levels of the sterile medium. We could therefore conclude that the source of the photon emission would be affected by genetic activity and is oxygen related.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Molecular phylogeny of theChironomus genus deduced from nucleotide sequences of two nuclear genes,ssp 160 and the globin 2b gene

Two genes were employed to study phylogenetic relatedness of theChironomus species: the protein-coding, salivary gland-specificssp160 gene, and the globin 2b (gb2b) gene. By using PCR, it was demonstrated that all the 38Chironomus species analyzed possess thegb2b gene, while only 13 have thessp160 gene. Partial nucleotide sequences of the genes of 22 species were determined. The data obtained were employed to construct phylogenetic trees which appeared to be topologically similar and revealed five groups of phylogenetically closely related species. Combining the data obtained in the studies of nuclear and mitochondrial genes, a molecular-data-based scenario could be suggested for theChironomus genus evolution.     

Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori

Summary The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.     

The narcolepsy-associatedDRw15, DQw6, Dw2 haplotype has no uniqueHLA-DQA or -DQB restriction fragments and does not extend to theHLA-DP subregion

Almost all patients with cataplectic narcolepsy are DR2-positive. It has been suggested that thenon-DR2 allele/haplotype might not be neutral with respect to disease susceptibility. It has also been reported thatTaq IDQA andBam HI,Eco RI,Eco RV, andPst IDQB restriction fragments might differentiate between narcoleptic and healthy DR2-positive individuals. In the present study,HLA class II gene polymorphisms were investigated by restriction fragment length polymorphism (RFLP) analysis in 47 Swedish patients with cataplectic narcolepsy, 100 random controls, and DR2-associated homozygous cell lines. All patients hadTaq IDRBDQA-DQB patterns corresponding to theDRw15,DQw6, Dw2 haplotype. The non-DR2 haplotype was found to be neutral. This genotyped group of patients allows firm rejection of a recessive mode of inheritance and supports a dominant or additive model. NoDQA orDQB RFLPs were found that could differentiate between DR2-positive narcoleptics, DRw15,DQw6,Dw2-positive controls, orDw2-homozygous cell lines. No significantMsp IHLA-DP association was found. No linkage disequilibrium was observed between theDRw15,DQw6,Dw2 haplotype and alleles of theDP subregion in patients or controls. Thus, theHLA-D region-associated narcolepsy susceptibility gene may be located telomeric to theHLA-DP subregion. No RFLPs have been observed that can locate the narcolepsy susceptibility gene closer to theDQ than to theDR subregion.     

Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium,Chlorobium vibrioforme by homologous recombination

Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec ₅₅₁ subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.