Francis Gasser

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.

Methylophaga marina gen. nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs

After enrichment in a medium containing seawater and methanol, 42 methylotrophic strains were isolated. All of these strains were gram-negative, strictly aerobic, motile, rod-shaped organisms that required vitamin B12. None grew on methane or on complex nutrient media supplemented or not supplemented with NaCI. All but 2 strains grew on methanol, methylamine, and fructose, 17 strains grew on dimethylamine, and 10 strains grew on trimethylamine. Fructose was the only multicarbon compound tested that was used as a growth substrate. All 11 strains tested used the ribulose monophosphate pathway of carbon assimilation. Depending on the strain, methylamine was oxidized either through a methylamine dehydrogenase or through a methylglut-amate dehydrogenase. The mean guanine-plus-cytosine content of 33 strains was 43 mol%. Based on deoxyribonucleic acid-deoxyribonucleic acid hybridization, two related groups were identified among 11 strains examined. We propose a new genus, Methylophaga, with two species, Methylophaga marina (type species) and Methylophaga thalassica. There was no significant deoxyribonucleic acid hybridization between Methylophaga and the terrestrial obligate methanol utilizers tested. The type strains of M. marina and M. thalassica are strains ATCC 35842 (= NCMB 2244) and ATCC 33146 (= NCMB 2163), respectively.

Deoxyribonucleic Acid Homologies in the Genus Leuconostoc

Six deoxyribonucleic acid (DNA)-DNA homology groups within 45 strains of the genus Leuconostoc were distinguishable through DNA-DNA hybridizations with four 3H-labeled reference DNA preparations from type or reference strains of the various known named species. There is good correspondence between two of these DNA-DNA hybridization groups and the species L. lactis and L. paramesenteroides. Strains named L. mesenteroides are heterogeneous, comprising three different hybridization groups. One of these groups includes all the strains named L. dextranicum and L. cremoris. These results agree precisely with previous results from an immunological study of the two isofunctional enzymes, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and d-lactate dehydrogenase (EC 1.1.1.28), prepared from L. lactis NCDO 546 and L. mesenteroides NCDO 768 (ATCC 12291).

Genetic analysis of the streptomycin resistance encoded by Tn5

SummaryTwo different aminoglycoside phosphotransferases were separated in crude extracts of Methylobacterium organophilum (Tn5). One is the aminoglycoside phosphotransferase (3′) type II (APH(3′)II), the only aminoglyconside modifying enzyme expressed by Tn5 in E. coli; the other is a streptomycin phosphotransferase (SPH). Various deletion mutants of Tn5 were cloned into pBR322 (plasmids pX) and co-transferred by the plasmid pJB3J1 into M. organophilum where stable cointegrants were formed. By the analysis of the resulting phenotypes, the streptomycin resistance gene was located in a 1,000 bp segment of the central region of Tn5. This gene is not expressed in E. coli. The possibility that this resistance gene is the structural gene of the SPH, is discussed. The formation of the cointegrants (pJB3J1; pX) is mediated through the IS21 sequence of pJB3J1. From two cointegrants KmR SmR in M. organophilum (pPM111 and pPM113), mutations KmS SmR and Kmr SmS were isolated. Restriction analysis of these mutants showed that the direction of transcription of the kanamycin and of the streptomycin resistance genes is identical. The existence of a particular promoter for the streptomycin resistance gene could not be firmly established because IS21 might have a promoter activity. However the postulated promoter was located toward the 3′ end of the APH(3′)II (Km) gene.

Brochothrix campestris sp. nov.

Two deoxyribonucleic acid hybridization groups were found among Brochothrix strains isolated from various sources. One genospecies contained 165 strains and included the type strain of Brochothrix thermosphacta. The other genospecies (five strains) represented a new species, for which the name Brochothrix campestris sp. nov. is proposed. The type strain is strain S3 (= CIP 102920 = ATCC 43754). B. campestris differs from B. thermosphacta by growth in the presence of 8 and 10% NaCI and 0.05% (wt/vol) potassium tellurite, by hippurate hydrolysis, and by acid production from rhamnose.

Mutants of Escherichia coli producing pyrroloquinoline quinone.

In glucose minimal medium a PTS- strain of Escherichia coli [delta (ptsH ptsI crr)] could grow slowly (doubling time, d = 10 h). When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population. These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation. They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ. Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium. Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain. Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism. The regulation of PQQ production by E. coli is discussed.

Comparative Immunological Relationships of Two Distinct Sets of Isofunctional Dehydrogenases in the Genus Leuconostoc

D-Lactate dehydrogenase (D-LDH) and glucose-6-phosphate dehydrogenase (G-6-P-DH), both purified from Leuconostoc lactis strain NCDO 546, were used to prepare specific antisera. Cross-reactions of varying intensity with the anti-D-LDH were obtained with all of the Leuconostoc strains and with certain strains of the heterofermentative lactobacilli studied. Cross-reactions with the anti-G-6-P-DH were obtained with all of the Leuconostoc strains except those of L. oenos; none of the heterofermentative lactobacilli cross-reacted. Pairwise comparisons between cross-reacting extracts with respect to each antiserum permitted the assignment of strains to groups of identical immunological specificity: with the exception of a single strain, the groups of strains revealed by the use of each antiserum coincided.

Immunological Relationships of Glucose-6-Phosphate Dehydrogenase of Leuconostoc mesenteroides NCDO 768 (= ATCC 12291)

An antiserum against the purified glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G-6-P-DH) of Leuconostoc mesenteroides NCDO 768 has been prepared. Cross-reactions were observed with extracts of other strains of Leuconostoc, comprising named strains of L. lactis, L. mesenteroides, L. dextranicum, L. cremoris, and L. paramesenteroides. Cross-reactions were not obtained with L. oenos. The groups of identical specificity and their order of decreasing similarity toward the homologous enzyme were determined, and the results are consistent with those obtained previously with another G-6-P-DH antiserum from L. lactis NCDO 546. Thus, except for L. oenos, many strains of Leuconostoc comprising multiple (three or more) species might be considered as phenotypically diverse but genetically rather uniform in their line of descent from a common ancestor.

Expression of Tn5-encoded streptomycin resistance in E. coli.

SummaryFour Tn5 mutations able to express streptomycin resistance in E. coli were obtained independently. These mutations (called Tn5*) were localized and sequenced. All of them consist of a 6 bp deletion in the str gene near the 3′ end. The mutation affects a region peculiar for its repetition of an identical 6 bp sequence. The mutation does not affect the level of transcription of the kan, ble, str operon of Tn5, neither does it increase the level of translation of str. The mutation seems to interfere with a post-translational event.

Séparation et purification de la D-lactico-déshydrogénase et de la glucose-6-phosphate déshydrogénase de Leuconostoc lactis Etude de quelques propriétés

Summary The NAD dependent D-lactic acid dehydrogenase and the NAD(P) dependent glucose-6-phosphate dehydrogenase from Leuconostoc lactis have been purified from the same crude extact by chromatography on hydroxyapatite. The two enzymes were then separately purified until obtention of fractions producing a single band of protein in polyacrylamide gel electrophoresis and a single line of precipitation by immunoelectrophoresis. The main properties of the two enzymes were studied and compared to the properties of isofunctionnal enzymes of other lactic acid bacteria. The D-lactic acid dehydrogenase of Leuconostoc lactis shows the same physical and kinetic properties than the Lactobacillus enzymes. These properties are quite different from those of other known bacterial D-lactic acid dehydrogenases.