- Volume 138, Issue 2, 1992

SUMMARY: The Salmonella enterica group C1 O antigen structure has a Man-Man-Man-Man-GlcNAc backbone with a glucose branch, which differs from the S. enterica group B O antigen structure which has a Man-Rha-Gal backbone with abequose as side-chain. We have cloned the group C1 rfb (O antigen) gene cluster from serovar montevideo strain M40, using a low-copy-number cosmid vector. The restriction map of the group C1 (M40) rfb gene cluster was compared with that of group B strain LT2 by Southern hybridization and restriction enzyme analysis. The results indicate that the flanking genes are very similar in the two strains, but there is no detectable similarity in the rfb regions. We localized the mannose pathway genes rfbM and rfbK and one of the genes, rfbK, shows considerably similarity to cpsG of strain LT2, suggesting that part of the mannose pathway in the group C1 rfb cluster is derived from a gene of the M antigen (cps) cluster. The M antigen, which forms a capsule, is comprised of four sugars, including fucose. The biosynthetic pathway of GDP-fucose has steps in common with the GDP-mannose pathway, and the cps cluster has isogenes of rfbK and rfbM, presumably as part of a fucose pathway. We discuss the structure and possible evolution of the group C1 rfb gene cluster.

SUMMARY: Previously we reported [Deane, S. M., Maharaj, R., Robb, F. T. & Woods, D. R. (1987) Journal of General Microbiology 133, 2295-2302] that the production of a Vibrio alginolyticus SDS-resistant alkaline serine protease (Pro A) cloned in Escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active Pro A. Replacement of the V. alginolyticus promoter region by the α-amylase promoter region from Bacillus amyloliquefaciens resulted in the simultaneous synthesis and secretion of Pro A in E. coli. The V. alginolyticus proA gene cloned on a shuttle vector did not produce active Pro A in Bacillus subtilis. Although Pro A has a typical Gram-positive signal sequence, it was not functional in B. subtilis. Replacement of the Pro A signal sequence with the α-amylase signal sequence resulted in the production of active Pro A in B. subtilis.

SUMMARY: A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2μ-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir ⚬) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir ⚬ host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.

SUMMARY: We have selected for genes that, when present in multiple copies, enhance growth of wild-type cells of Saccharomyces cerevisiae in an iron-limiting medium. A gene designated FUP1, for ‘ferric utilization proficient’, was isolated by this approach. Increased dosage of FUP1 reduces the concentration of iron in the medium required for efficient growth and confers elevated levels of iron uptake activity in iron-limited cells. Disruption of the FUP1 locus reduces wild-type iron uptake rates by 2-fold in cells grown on raffinose medium but has no effect on glucose-grown cells. DNA sequencing showed that FUP1 encodes a hydrophilic 43 kDa protein identical to MSN1, a gene encoding a transcriptional activator implicated in carbon source regulation. Our results suggest that FUP1/MSN1 also regulates synthesis of gene products involved in iron uptake.

SUMMARY: The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6·0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

SUMMARY: Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9·7%. The molecular mass of the purified mutacin preparation was estimated to be 6·5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 °C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by α-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.

SUMMARY: The two 4·6 kb chloramphenicol resistance (CmR) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed M r values of 70000 on FPLC and were composed of three identical subunits, each of M r approximately 23000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5·7 and that of the CAT from pSCS7 pH 5·2. Both CAT variants exhibited highest enzyme activities at pH 8·0. The K m values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2·5 μM and 58·8 μM, respectively, while those of the CAT from pSCS7 were 2·7 μM and 55·5 μM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.

SUMMARY: The antigenic relationships of the iron-regulated proteins (IRPs) in Pasteurella haemolytica A and T biotype strains were examined by SDS-PAGE and immunoblotting. P. haemolytica cells of the A biotype, grown under conditions of iron-limitation, expressed two IRPs, of 35 and 70 kDa. All T biotype strains expressed IRPs with slightly different molecular masses of 37 and 78 kDa. Immunoblotting of all 16 P. haemolytica serotypes was carried out using a panel of polyclonal and monoclonal antibodies raised against serotype A2 antigens. Polyclonal antibodies revealed inter-serotype cross-reactivity towards the 35 and 70 kDa IRPs within the A biotype but no cross-reactivity against a T biotype protein in the 78 kDa region. Monoclonal antibody against the 35 kDa antigen reacted only with the A biotype 35 kDa IRP. Identical profiles were obtained for 10 field isolates of serotype A2, further emphasizing the antigen conservation within the A biotype. These findings reinforce the view that the A and T biotypes of P. haemolytica should be considered as separate species and suggest that IRPs from single A and T biotype strains incorporated into a vaccine might provide cross-protection against all P. haemolytica serotypable strains. Similar studies on the IRPs of 10 untypable strains revealed some of these to have different antigenic reactivities from those observed within the A and T biotypes.

SUMMARY: A turbidostat culture technique was used to study the effects of different salt shocks on the freshwater cyanobacteria Synechocystis sp. strain PCC 6803 and Microcystis firma. Shocks were performed either suddenly or gradually, on both unacclimated cultures and those pre-acclimated to 0·77 M-NaCl. All suddenly shocked cultures exhibited a decline in growth after a few hours, characterized by severely decreased metabolic activities (e.g. photosynthesis, respiration, glucose-6-phosphate dehydrogenase activity) and a time course of restoration which coincided with the accumulation of glucosylglycerol. Additionally, all untreated cultures had a late (after a few days) growth depression, distinguished by the stagnation of cell division. This was overcome by physiological adaptation of the whole cells or selection of cells with superior salt tolerance. The different types of growth depressions and the unique pattern of glucosylglycerol accumulation led to the conclusion that glucosylglycerol was necessary to maintain metabolic processes, but that this alone cannot account for successful salt acclimation.

SUMMARY: Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to Nα-acetyl-L-tryptophan.

SUMMARY: Partial 16S ribosomal ribonucleic acid sequences of five Aerococcus-like organisms originally isolated from patients with urinary tract infections were determined using reverse transcriptase in order to clarify their taxonomic position. Analysis of the sequence data revealed that the clinical isolates represent a hitherto unknown line of descent within the genus Aerococcus. A new species, Aerococcus urinae, is proposed for these isolates. The type strain is NCFB 2893.