- Volume 142, Issue 9, 1996

The high-affinity galactose permease, which comprises the periplasmic galactose receptor MgIB, the membrane translocator MgIC and the membrane-associated ATPase MgIA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli. This reduced transport activity correlated with a reduction in the quantity of MgIB. At 42 °C, an accumulation of pre-MgIB in the dnaK temperature-sensitive mutant reflected a defect in MgIB export. In addition, an accumulation of pre-MgIB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor. At 30 °C, there was no accumulation of pre-MgIB in the dnaK mutant, but there was still a decreased amount of MgIB in the periplasm. The reduction in MgIB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MgIA protein (which is expressed from the same operon as MgIB) was synthesized in normal amounts. Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mgIBAC mRNA, and a more stable and more abundant mgIB mRNA, produced by 3′-5′ degradation of the mgIBAC mRNA (R. W. Hogg, C. Voelker & I. von Carlowitz, 1991, Mol Gen Genet 229, 453–459). The mgIB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3′ extremity, which is responsible for the higher expression of MgIB compared to MgIA and MgIC. The decreased MgIB expression in the dnaK mutant at 30 °C in the present work correlated with a reduced stability of the mgIB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mgIB gene.

Achromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. Nothing is known concerning its phylogeny and it has eluded all attempts at laboratory cultivation. The limited physiological description of A. oxaliferum has been based on morphological features of the bacterium such as the presence of intracellular sulphur inclusions. A. oxaliferum cells were purified from a wetland region close to Rydal Water (Cumbria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sulphur inclusions within the cells, were present in a single sample of purified cells. PCR was used to amplify almost full-length 16S rRNA gene sequences from DNA extracted from A. oxaliferum cells directly purified from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three different sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a representative clone of the most dominant sequence-type (52 % of the sequences). Comparative sequence analysis showed A. oxaliferum to form a deep branching lineage within the γ-subdivision of the Proteobacteria. A. oxaliferum was related most closely to the Chromatium assemblage that includes sulphur-oxidizing symbiotic bacteria, purple sulphur bacteria, and sulpur- and iron-oxidizing thiobacilli. Phylogenetic inferences made using distance, parsimony and maximum likelihood methods all placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum cells however formed a monophyletic group well supported by bootstrap analysis (85–100% support depending on the analysis done). It was concluded that A. oxaliferum was related to organisms of the Chromatium assemblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequence obtained, by the use of a fluorescently-labelled 165 rRNA-targeted oligonucleotide specific for the A. oxaliferum rRNA sequence.

Co-adhesion between oral microbial pairs (i.e. adhesion of a planktonic micro-organism to a sessile organism adhering to a substratum surface) has been described as a highly specific interaction, mediated by stereochemical groups on the interacting microbial cell surfaces, and also as a non-specific, critical colloid-chemical interaction. In a colloid-chemical approach, microbial co-adhesion is considered as an interplay between, amongst others, hydrophobic and electrostatic interactions. The aim of this paper was to determine the influence of ionic strength on the co-adhesion of Streptococcus oralis 34 to either Actinomyces naeslundii T14V-J1 or its mutant strain 5951 adhering to glass in a parallel-plate flow chamber. To this end, the ionic strength of the suspension was varied by the addition of KCl. Another aim was to investigate whether substratum hydrophobicity affected the co-adhesion between the organisms by allowing the sessile organisms (in this case the actinomyces) to adhere either to hydrophilic or to hydrophobic, dimethyldichlorosilane (DDS)-coated glass. The kinetics of co-adhesion of S. oralis 34 to the actinomyces decreased with increasing ionic strength, expressed as the ratio, χ, between the local and non-local initial deposition rates of the streptococci in the vicinity of, or far away from, the adhering actinomyces, respectively. In a stationary end-point of co-adhesion, ionic strength appeared not to be a determinant factor for the co-adhesion of S. oralis 34 with A. naeslundii 5951, either when the actinomyces were adhering to hydrophilic glass or to hydrophobic, DDS-coated glass. However, for S. oralis 34 co-adhering in a stationary end-point with A. naeslundii T14V-J1 in the high-ionic-strength (250 mM KCl) suspension, co-adhesion was far less on hydrophobic, DDS-coated glass than on hydrophilic glass. It is possible that the hydrophobic fibrils on A. naeslundii T14V-J1 bearing the lectin responsible for co-adhesion were immobilized in the latter case by adsorption to the hydrophobic substratum, making them less available for interaction with the streptococci.

Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this slp segment. In this study the presence of S-protein and corresponding S-protein-encoding genes of strains belonging to species that are closely related to L. acidophilus was determined. All strains investigated were identified by numerical comparison of highly standardized one-dimensional SDS-PAGE whole-cellular-protein patterns. Western blot and Southern blot methods were used to identify the presence of, and homology between, S-proteins and S-protein-encoding genes. From these analyses we conclude that strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum possess an S-layer and contain two slp genes. Strains of L. helveticus possess an S-layer but have only one intact slp gene. Strains of L. gasseri, L. johnsonii and L. delbrueckii subsp. bulgaricus have neither an S-layer nor S-protein-encoding genes hybridizing with probes derived from the L. acidophilus slpA or slpB region. The presence of a highly conserved 5′ region in the slp genes of strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum suggests that S-layer variation is a common feature for strains of these species.

The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis. Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component. Changes throughout the molecule also affected its induction capacity. The production of certain variant nisins by engineered lactococcal strains is reduced in paralle with the strains’ immunity to nisin. This can be attributed to inefficient induction by the variant molecule. Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains.

F-like plasmid TP181 is particularly susceptible to curing by the naphthoquinone derivative plumbagin, which may attack DNA gyrase. TP181 should provide particular insight into the basis of plasmid elimination, which has application in a number of contexts. Curing was found to be optimal at pH 7·2. An EcoRl fragment containing the RF1A replicon of TP181 was joined to a KmR determinant (giving miniTP181). MiniTP181 had the same increased susceptibility to curing by plumbagin when compared to miniF as TP181 had relative to F. Plumbagin interfered with replication of miniTP181, depressing its copy number and increasing the rate of segregation. Plumbagin also blocked the lethal effect of the ccd locus after rifampicin treatment, which mimics production of plasmid-free segregants, so that more of these plasmid-free cells would survive. Restriction mapping and DNA sequence analysis indicated that the ccd locus of TP181 is almost identical to that of F but that TP181 lacks the repC gene present in F which is needed to activate replication from oriV. Thus the sensitivity of TP181 may be due to its dependence on both a replicon which is hypersensitive to perturbation of supercoiling by plumbagin and a host-killing system which is blocked by plumbagin.

We have determined the entire nucleotide sequence of a 4·4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701. Two complete ORFs were identified and termed mopA and mopB. mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes. Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein. An assay was developed to demonstrate MopB function in substrate uptake. Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition. The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD. Northern hybridization experiments determined that mopA and mopB are cotranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate. mopA and mopB may be situated at the 3′-terminus of a cistron about 10 kbp in size. The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities.

The promoter of the gua operon has been located by transcript mapping using primer extension with reverse transcriptase. The surrounding nucleotide sequence has features characteristic of promoters under stringent and growth-rate-dependent regulation, namely a GC-rich discriminator next to the − 10 hexamer, an upstream AT-rich sequence (the UP element) and potential FIS-binding sites. Transcriptional activity of the gua promoter was examined using transcriptional fusions to lacZ placed at a single chromosomal location. Expression from gua was reduced under stringent conditions in vivo, and varied with growth rate. Growth-rate control was independent of guanine-mediated repression. A fusion in which the GC-rich discriminator was mutated by insertion of an AT-rich oligonucleotide was used to demonstrate the importance of this region in control. Both stringent and growth-rate-dependent controls were abolished by the mutation. Other potential regulatory signals in the vicinity of the gua promoter are a pur operator (binding site for the PurR repressor), a gua operator, a DnaA-binding site and a CRP/FNR-binding sequence. The gua promoter lies back-to-back with the promoter for xseA (exonuclease VII), the two promoters being separated by only 20 bp.

Genes from the Mycobacterium tuberculosis purine biosynthetic pathway were identified using purine auxotrophic mutants of Mycobacterium smegmatis obtained by Tn 611 transposon mutagenesis. Two approaches were followed in parallel. The first consisted of the complementation of the M. smegmatis purine auxotrophs using a M. tuberculosis H37Rv shuttle cosmid library. In the second approach, specific probes corresponding to the regions adjacent to the insertion sites of Tn 611 in the M. smegmatis genome were used to screen a M. tuberculosis plasmid library by colony hybridization for inserts carrying homologous DNA fragments. Nucleotide sequence analysis of two M. tuberculosis genes isolated by these methods revealed high similarities with purC and purL genes from other bacterial and fungal sources. Transcriptional start sites were mapped for both genes, which revealed similar −10 boxes but with a higher GC content than the Escherichia coli σ70 consensus.

We have found a new insertion sequence (IS), designated IS Aa1, downstream of the S10 operon in Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4. IS Aa1, the first IS element characterized in this organism, is 705 bp long and lacks terminal inverted repeats. This element displayed significant homology with IS200. Hybridization patterns of genomic DNA of seven A. actinomycetemcomitans strains with an internal ISAa1 probe varied depending on the serotypes, suggesting that IS Aa1 might be a useful tool for epidemiological studies.

An insertion sequence (IS) of Mycobacterium xenopi has been isolated and sequenced. This 1323 bp element, designated IS1395, is present in up to 18 copies in the M. xenopi genome and may be harboured in an M. xenopi extra-chromosomal element. It encodes a putative transposase of 415 amino acids which displays sequence homology to the Staphylococcus aureus IS256 family. Members of this class of elements have been described in the genus Mycobacterium - for example IS1081 is present in the M. tuberculosis complex, IS1245-IS1311 in M. avium, and IS6120 in M. smegmatis; these elements exhibit an 89%, 45% and 16% amino acid identity with IS1395, respectively. Investigation of the host range of IS1395 by Southern blot analysis revealed additional IS1395-related repeated sequences in M. gordonae and M. celatum. Moreover, IS1395 represents a useful epidemiological tool for M. xenopi strain typing as it provides a diversity of restriction fragment length polymorphism patterns.

Linear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi. All bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes. We found a single gene in B. burgdorferi (named hbb) whose product (named Hbb) complements the defects for λ DNA packaging found in E. coli strains mutant in the genes for IHF and HU. The sequence of the predicted B. burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria. The gene appears to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E. coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function. This operon is located upstream of the previously identified B. burgdorferi rho homologue.

A 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae λ phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful.