Association of enterohemolysin and non-fermentation of rhamnose and sucrose with Shiga-like toxin genes in Escherichia coli from calves.

Fecal Escherichia (E.) coli strains from calves were tested for simply detectable phenotypical features associated with Shiga-like toxin (SLT) genes. DNA hybridization with SLT-specific oligonucleotide gene probes (detection of genes for SLT-I and SLT-II) was the "gold standard" for the evaluation of Vero cell cytotoxicity, fermentation of several saccharides, beta-D-glucuronidase activity and production of alpha-hemolysin (alpha-Hly) or enterohemolysin (E-Hly). While SLTEC and non-SLTEC did not significantly differ in production of alpha-Hly, beta-D-glucuronidase activity and fermentation of D-sorbitol, production of E-Hly and non-fermentation of L-rhamnose (Rha) and D-sucrose (Suc) were associated with SLT genes. Sensitivity and specificity of the E-Hly+ phenotype were 53% and 88% for identification of calf SLTEC. When three markers were combined to form the parameter ["E-Hly+ or (Rha- and Suc-)"], sensitivity was higher (65%) and specificity was almost the same (85%). Production of enterohemolysin and inability to ferment rhamnose and sucrose were more often associated with the SLT-I gene than with SLT-II genes. Approximately 71% SLT-I+ E. coli were positive in the enterohemolysin assay. The test combination "E-Hly+ or (Rha- and Suc-)" and Suc-)" was most valuable for the presumptive identification of SLT-I+ E. coli (sensitivity 85%, specificity 83%). These data suggest that the phenotype "E-Hly+ or (Rha- and Suc-)" may be a helpful marker for the detection of SLT-I+ E. coli in SLTEC associated diarrhoea of calves.

Distribution and evolution of nisin-sucrose elements in Lactococcus lactis.

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.

Transcriptional regulation of the Tn5276-located Lactococcus lactis sucrose operon and characterization of the sacA gene encoding sucrose-6-phosphate hydrolase.

The Lactococcus lactis sucrose operon was located on the conjugative transposon Tn5276 and the nucleotide sequence of the sacA gene, encoding sucrose-6-phosphate hydrolase, and its surrounding regions was determined. Northern blot analysis showed that the sucrose operon contains two divergent transcriptional units of 3.2 and 3.6 kb, the expression of which is considerably higher in cells grown on sucrose than in cells grown on glucose. This was confirmed by primer extension studies which demonstrated that transcription is initiated at two sucrose-inducible promoters with a back-to-back organization. The 3.2-kb transcriptional unit includes the sacB gene which most probably encodes the sucrose-specific enzyme II of the phosphotransferase system, and may contain the gene encoding fructokinase. The 3.6-kb transcriptional unit includes genes sacA and sacR. The protein encoded by the sacR gene is likely to be involved in the regulation of the sac operon expression, since its deduced N terminus is homologous to helix-turn-helix DNA-binding domains found in several regulatory proteins.

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.

Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.

Evidence that Candida stellatoidea type II is a mutant of Candida albicans that does not express sucrose-inhibitable alpha-glucosidase.

Candida stellatoidea is classically distinguished from C. albicans by the ability of the latter species to assimilate sucrose. We show here that sucrose-positive revertants of C. stellatoidea type II are readily isolated and that C. stellatoidea type II strains probably resulted from a mutation in the sucrase gene of C. albicans. The revertants were not laboratory contaminants, as determined by restriction fragment length polymorphism analysis and retention of an auxotrophic marker. The reversion of three tested strains was accompanied by 16 to 110-fold increases in expression of a sucrase/alpha-glucosidase but not an invertase, with a Km for sucrose of about 1 mM. The enzyme activity was assayable in intact cells. The drastically increased expression of such an enzyme would allow extracellular sucrose hydrolysis and assimilation of the monosaccharide products.

[Structural and functional organization of the transposon Tn2555 carrying saccharose utilization genes]. / Strukturnaia i funktsional'naia organizatsiia transpozoma Tn2555, nesushchego geny utilizatsii sakharozy.

Transposon Tn2555 was isolated from a clinical E. coli strain carries the genes for sucrose utilization. Previously it was shown that Tn2555 is very unstable and undergoes structural rearrangements with a high frequency. Several deletion derivatives of Tn2555 and one with an inversion of the internal segment were found. They form the Tn2555 transposon family. This paper describes further structural and functional analysis of Tn2555. In the course of the experiments on pBR325 (Mob-) mobilization by conjugative RP4 derivatives, containing Tn2555 family elements, it was found, that all of them induce cointegrate formation. Some of these cointegrates were able to dissociate in rec+ and recA E. coli cells. Restriction endonuclease analysis of the resulting plasmids have shown, that among them were the end products of the Tn2555 transposition from RP4 to pBR325. Besides, the pBR325 derivatives, containing a discrete DNA segment of approximately 800 b.p., originating from Tn2555, were found. The segment can transpose from pBR325 to RP4 indicating that it is an insertion sequence. This new IS-element was designated IS286. The size and the genetic properties of IS286 resemble those of the IS1 element. However restriction analysis and Southern hybridization data show no significant homology between IS286 and IS1. It was found that the Tn2555 family elements are flanked by directly repeated IS286. One of them (Tn2555.3) contains an additional copy of IS286 in its internal region.