A 56-kb plasmid was identified in Lactococcus lactic subsp. lactis (L. lactis) M189 which encodes resistance to nisin (NisR) following mobilization of the plasmid into L. lactis LM0230. The NisR determinant was localized on a 1.6-kb Hindlll fragment by DNA restriction fragment deletion and subcloning. An open reading frame (ORF) of 957 bases was identified by sequence analysis and its transcription start site was mapped by primer extension. The ORF is flanked by two regions which exhibit complete homology to parts of the inverted repeat sequences of IS981 and ISS1T. The promoter for transcription was found to consist of an extended -10 site (TgTGtTATAAT) that lacks a -35 site. Function of the extended -10 promoter was demonstrated by its ability to express the promoterless cat gene from Staphylococcus aureus. Base substitution analysis revealed that the TgTGt extension is essential for promoter efficiency in L. lactis.
A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb Xbal fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.
Mixed rumen bacteria, isolated by centrifugation from the rumen of steers fed a roughage (R) or concentrate (C) diet, were used to determine if lectins are present on rumen bacteria, based on hemagglutination (HA) and HA inhibition assays in vitro. Rumen bacteria from steers fed either diet agglutinated erythrocytes from cattle, sheep, pigs, and rats, suggested that lectins exist on rumen bacteria. Bacterial HA titers from steers receiving the R diet were much higher (p<0.001) than those from steers fed the C diet, depending on the erythrocyte source used. Centrifugation at 20, 000×g for 30min fractionated the rumen bacteria into upper(U)and lower(L)layers. The HA titers of the U bacterial fractions were significantly higher (p<0.001) than those of the L fractions. A remarkable reduction or complete disappearance of HA titers following treatment of rumen bacteria with protease, trypsin, sodium dodecyl sulfate (SDS) or sodium periodate indicates that rumen bacterial lectins are probably glycoproteins. Lectin specificity for saccharides (galactose, lactose, N-acetyl-D-galactosamine, methyl-α-D-galactopyranoside and methyl-β-galactopyranoside) and glycoproteins (mucin, fetuin, and thyroglobulin) was found in the RU, RL, and CU bacterial fractions; no specific binding was determined in the CL fractions. The potential role of lectins in mediating the attachment of rumen bacteria to feed particles, rumen epithelia and other microorganisms is discussed.
16S ribosomal RNA gene sequences from seven strains of Aquaspirillum peregrinum, Aqu. itersonii, Aqu. Polymorphum, and Oceanospirillum pusillum were compared with homologous sequences from other members of helical-shaped bacteria. The bootstrapped neighbor-joining tree, inferred from 887 aligned sites, placed the spirillum taxa assigned to Aquaspirillum, Oceanospirillum, Azospirillum, Magnetospirillum, Rhodospirillum, and Rhodocista of the Proteobacteria in seven clusters of α Proteobacteria separately from other shapes of bacteria. Aqu. Peregrinum and Aqu. Itersonii grouped together in 88% bootstrap support. They were more related to Rhodospirillumrubrum and Rsp. Photometricum than Aqu. polymorphum. Aqu. Polymorphum was close to Magnetospirillum gryphiswaldense, Mag. magnetotacticum, Rsp. fulvum, and Rsp. Molischianum, and more close to Mag. gryphiswaldense. Oce. Pusillum was not related to other spirillum taxa and was placed in a separate branch. Rhodocista was very closely related to Azospirillum. Photosynthesis and magnetotaxis, as phenotypic characters, were not important in the classification of helical bacteria.
Two new strains, Pseudomonas sp. TCP114 degrading 2, 4, 6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.
Acinetobacter sp. strain ST-1, isolated from garden soil, can mineralize 4-chlorobenzoic acid (4-CBA). The bacterium degrades 4-CBA, starting with dehalogenation to yield 4-hydroxybenzoic acid (4-HBA) under both aerobic and anaerobic conditions, suggesting that the dehalogenating enzyme in the strain is not an oxygenase; the enzyme may catalyze halide hydrolysis. To identify the oxygen source of the C4-hydroxy groups in the dehalogenation step, we used H218O as the solvent under anaerobic conditions. When resting cells were incubated in the presence of 4-CBA and H218O under a nitrogen gas stream, the hydroxy group on the aromatic nucleus of the 4-HBA produced was derived from water, not from molecular oxygen. This dehalogenation was hydrolytic, because analysis of the mass spectrum of the trimethylsilyl derivative of one of the metabolites, 18O-labeled 4-HBA, showed that 80% of the C4-hydroxy groups were labeled with 18O. Hydrolytic dehalogenation of 4-CBA in intact cells has not been reported earlier. To identify substrate specificity, we next examined the ability of the strain to dehalogenate 4-CBA analogues and dichlorobenzoic acids. The results of metabolite analysis by high-pressure liquid chromatography showed that the strain dehalogenated 4-bromobenzoic acid and 4-iodobenzoic acid, yielding 4-HBA, suggesting that these compounds could be further degraded and mineralized by the strain via the β-ketoadipate pathway, as occurs with 4-CBA. This strain, however, did not dehalogenate 4-fuorobenzoic acid, 2- and 3-chlorobenzoic acids, or 2, 4-, 3, 4-, and 3, 5-dichlorobenzoic acids during 4 days of incubation, implying that the dehalogenating enzyme of the strain has high substrate specificity.
An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B6 (pyridoxine hydrochloride) (B6) on the production of lysine from the three stereoisomers of 2, 6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12h with and without DAP-SI (5mM) as a substrate and SL (5μg/ml) and/or B6 (10μg/ml) as additives. In P suspensions, SL and B6 reduced the amount of DAP-SI by 2.1 times (p<0.001, where p is probability) and 19.9% (p<0.05), respectively, and also increased the production of lysine by 2.4 times (p<0.001) and 26.8% (p<0.05), respectively, during 12h incubation. In B suspensions, the reductions of DAP-SI with a single addition of SL or B6 were 8.5% (p<0.001) and 2.7%, respectively, and lysine production increased by 54.3 and 32.9% (p<0.001), respectively, during 12h incubation. In PB suspensions, the reductions of DAP-SI were 21.9 and 11.7% (p<0.001) with a single addition of SL or B6, respectively, and the production of lysine increased by 81.4 and 39.4% (p<0.001), respectively, during 12h incubation. When SL and B6 were added together to the P, B, and PB suspensions, lysine production further increased by 12.3, 21.3, and 12.4% more than the cases of adding SL only during 12h incubation, respectively. SL and B6 were demonstrated to enhance the production of lysine from DAP-SI by mixed rumen protozoa, mixed rumen bacteria and their mixture in this study.