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Sakacin P is a small, heat-stable, ribosomally synthesized peptide produced by certain strains of Lactobacillus sake. It inhibits the growth of several Gram-positive bacteria, including Listeria monocytogenes. A 7.6 kb chromosomal DNA fragment from Lb. sake Lb674 encompassing all genes responsible for sakacin P production and immunity was sequenced and introduced into Lb. sake strains Lb790 and Lb706X which are bacteriocin-negative and sensitive to sakacin P. The transformants produced sakacin P in comparable amounts to the parental strain, Lb674. The sakacin P gene cluster comprised six consecutive genes: sppK, sppR, sppA, spiA, sppT and sppE, all transcribed in the same direction. The deduced proteins SppK and SppR resembled the histidine kinase and response regulator proteins of bacterial two-component signal transducing systems of the AgrB/AgrA-type. The genes sppA and spiA encoded the sakacin P preprotein and the putative immunity protein, respectively. The predicted proteins SppT and SppE showed strong similarities to the proposed transport proteins of several other bacteriocins and to proteins implicated in the signal-sequence-independent export of Escherichia coli haemolysin A. Deletion and frameshift mutation analyses showed that sppK, sppT and sppE were essential for sakacin P production in Lb706X. The putative SpiA peptide was shown to be involved in immunity to sakacin P. Analogues of sppR and spiA were found on the chromosomes of Lb. sake Lb706X and Lb790, indicating the presence of an incomplete spp gene cluster in these strains.

The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme.

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)