The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.
The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M r of 76725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M r of the mature form is 75000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676–1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.
To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable Bacillus subtilis diploid cells through protoplast fusion. Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains. The ploidy of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization. Within the precision of these methods, all colonies were found to consist of haploid cells. A previously described non-complementing diploid was also found to be haploid. Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding. Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e. their plating within a distance of about 0·4 mm. The previously reported experimental results can be accounted for in the light of our results.
A prime plasmid has been used as the basis for the construction of a physical and genetic map of a 125 kb segment of the Pseudomonas aeruginosa PAO chromosome. Using pMO1811, a prime plasmid selected for the catA region, a series of Tn5 insertions were obtained which identified two new markers gcu (glycine utilization) and oap (organic acids and alcohols permeability) in the 125 kb region and located them in relation to other known markers of this region. A cosmid bank was constructed from the prime plasmid and an ordered array of cosmid clones for this region identified by restriction endonuclease mapping with EcoRI, HindIII and KpnI, as well as complementation mapping and chromosome walking. By Southern hybridization analyses, it was confirmed that the chromosomal insert carried by pMO1811 was flanked by single, tandemly arranged copies of IS 21 and the orientation of the insert on this prime was determined. This cosmid bank provides a resource for the further analysis of this region of the P. aeruginosa genome.
The precise location of the rhaT gene, encoding rhamnose permease, has been established between sodA and rhaC at 3605–3607 kb of Kohara's physical map, which corresponds to 88·4 min on the Escherichia coli chromosomal map. The dependence of the activity of the rhaT product on the function of rhaC, the rhamnose operon regulatory gene, was established by measuring rhamnose transport in wild-type and rhaC-deficient strains. The sequence of the sodA−rhaC interval displayed a single ORF corresponding to rhaT, which is transcribed counterclockwise on the E. coli chromosome. The ORF was shown to be preceded by a ribosome binding consensus sequence and a catabolite repression protein consensus sequence. The derived amino acid sequence displayed very low homology with any other permease and was clearly dissimilar to the homologous group formed by the xylose, arabinose, galactose and several glucose transporters. Analysis of the rhaT primary sequence identified potential membrane-spanning regions, possibly defining a protein structure model different from the one corresponding to the above-mentioned homologous group.
The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs−lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations are cis-acting and that phs is not subject to autoregulation.
A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5·4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M r and N-terminal amino acid sequence as the purified subunit of BPO-A2.
A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11·5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.
The temperate bacteriophage mv4 is representative of a widespread phage genetic group of Lactobacillus delbrueckii subsp. lactis or bulgaricus. The genome of this phage is circularly permuted and terminally redundant, as shown by mature mv4 homoduplexes, Southern hybridization and restriction enzyme analysis. A circular map of the mv4 genome was established, with unique sequences totalling 36 kb. The genomic location of the pac site (packaging site of mv4 DNA into phage heads) and the att site (phage integration site into the host cell chromosome) was determined. The genes coding for the two main structural phage proteins and for a phage-associated lysin were also mapped. Phage mv4 is capable of transducing a limited set of pieces of bacterial DNA and several specific chromosomal attachment sites of phage mv4 were identified. Bacteria lysogenic for phage mv4 were shown to be immune to infection by L. delbrueckii virulent phages related to mv4.
An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G+C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G+C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.
Of the eight Candida albicans chromosomes, chromosome 2, assigned by the MGL1 probe, is more variable in size than the other chromosomes among strains. We found that the clonal variation of chromosome 2, which carries a rDNA gene, occurred at a frequency of up to 10% of the progeny clones. After total chromosomal digestion with XhoI, which has no recognition sites within the rDNA repeat unit, the fragments containing the rDNA cluster were detected by Southern hybridization. The difference in fragment sizes corresponded to the clonal size variation of chromosome 2. The intensity of hybridization with rDNA also correlated with the difference in size. In addition, there was no size change in the non-rDNA region as detected by NotI digestion of chromosome 2, and there was no observed change in the individual rDNA basic repeat unit size. From these lines of evidence, we confirmed that the clonal size variation of chromosome 2 which occurs at high frequency is derived from the size change of the rDNA cluster.
Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 β-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the β-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 β-lactamase.