- Volume 144, Issue 8, 1998

K+-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, orf1, whose function is not clear. Products of V. alginolyticus trkA and orf1 were detected in E. coli minicells. trkA and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli ΔtrkA and an E. coli ΔtrkG ΔtrkH strain, respectively, suggesting that these V. alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli ΔsapABCDF (ΔtrkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K+-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.

Aerial mycelium and hyphal strands of Agaricus bisporus, strain U1, exhibited a rodlet pattern at their surfaces characteristic for assembled class I hydrophobins. An SDS-insoluble/trifluoroacetic-acid-soluble fraction from strands was found to contain one abundant protein with an apparent molecular mass on gel of 19 kDa. Two sequences for this protein (ABH3), typical of class I hydrophobins, could be deduced by sequencing cDNA clones obtained by RT-PCR. The two forms of the protein could be assigned to different alleles present in the two homokaryons that constitute the heterokaryotic U1 strain. ABH3 displays all the in vitro properties of a typical class I hydrophobin such as SC3 from Schizophyllum commune but is not glycosylated or otherwise post-translationally modified because the molecular mass values deduced from the amino acid sequence (9228 and 9271 Da) and derived from mass spectrometry were in good agreement. The ABH3 transcript was found to be present in the vegetative mycelium of both primary and secondary mycelium but not in the fruiting bodies, whereas the reverse was found for the ABH1 hydrophobin. Using an S. commune mutant with a disrupted SC3 gene it was found that ABH3 can substitute for SC3 in inducing formation of aerial hyphae, suggesting a role of ABH3 in the emergence of aerial hyphae and strands in A. bisporus.

Bacillus thuringiensis subsp. israelensis IPS78 and B. thuringiensis subsp. aizawai HD133 both secreted exochitinase activity when grown in a medium containing chitin. Allosamidin, a specific chitinase inhibitor, inhibited activity from both strains, with IC50 values of about 50 μM with colloidal chitin as substrate and between 1 and 10 μM with 4-methylumbelliferyl-diacetylchitobioside and 4-methylumbelliferyl-triacetylchitotrioside as substrates. The involvement of these chitinolytic activities during pathogenesis in insects has been investigated with B. thuringiensis subsp. israelensis IPS78 against larvae of the midge Culicoides nubeculosus, and with B. thuringiensis subsp. aizawai HD133 against caterpillars of the cotton leafworm Spodoptera littoralis. Presence of 100 μM allosamidin increased the LD50 by factors of 1.3 and 1.4, respectively, demonstrating a role for bacterial chitinases in the attack on the insects. Presence of chitinase A from Serratia marcescens considerably decreased the values for LD50' confirming previous observations with different systems of the potentiation of entomopathogenesis of B. thuringiensis by exogenous chitinases. The most likely action of the endogenous chitinases of B. thuringiensis is to weaken the insects' peritrophic membranes, allowing more ready access of the bacterial toxins to the gut epithelia. Addition of exogenous chitinases will then increase this effect. Complementary cross-infection experiments, strain HD133 against midge larvae and strain IPS78 against caterpillars, were performed to investigate the pathogen/host specificities of the effects. Results showed that much higher concentrations of bacteria were required to achieve even low mortalities, and addition of chitinase A gave no increase in death rate.

Escherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. The isolates were characterized for the production of colicins and their resistance to the co-occurring colicins. Of the 447 isolates examined, 59% were found to be colicinogenic. Phenotypic and PCR-based genotypic methods were used to determine the types of colicins being produced. Colicin E2 was the most common, representing 27% of the colicin-producing isolates. Colicin la was produced by 3% of the colicinogenic isolates. The remaining colicins could not be identified, but phenotypic and PCR data argue that at least nine different colicin types are present in this collection of E. coli. The frequency of colicinogenic isolates declined from 71% to 43% over the 7 months of the study. All colicin types appeared to decline in frequency. Concurrently, the resistance of isolates to colicin E2 increased from about 50% to 70%. Two hypotheses are proposed to explain the decline in the frequency of colicinogeny in this population of E. coli. The first relates to the within-host interactions occurring among colicinogenic, colicin-susceptible and colicin-resistant populations within a host. The second relates to the among-host interactions between susceptible and colicinogenic populations and the effect of host population densities on these interactions.

The effect of manganous ions [Mn(II)] and ferrous ions [Fe(II)] on expression of photosynthesis genes in Rhodobacter sphaeroides was investigated. The presence of Mn(II) during phototrophic (anaerobic) and chemotrophic (aerobic) growth of R. sphaeroides caused a decrease in the amount of bacteriochlorophyll and carotenoid pigments which were synthesized and this was associated mainly with a decrease in the level of light-harvesting complex II. Mn(II) was shown to cause a decrease in expression of the puc operon, which encodes the polypeptides of light-harvesting complex II. Expression of the puc operon is controlled by the central repressor of photosynthesis gene expression, PpsR. In a ppsR mutant there was no effect of Mn(II) on photosynthesis gene expression. It is concluded that Mn(II) may act as a compressor in the action of PpsR or act via an as yet uncharacterized protein that interacts with PpsR. In contrast to the effects of Mn(II), Fe(II) was required for high levels of photosynthesis gene expression. This requirement for Fe(II) was shown to be related to the regulation of hemA, a gene under the control of the transcriptional regulator, FnrL. Mn(II) did not affect FnrL-dependent gene expression.

The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to ‘T-box’ leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U)AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16 · 3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; IpdA) is the distal gene of another cluster containing two promoters located at 2 · 7 min: Ppdh pdhR-aceEF-PIpd IpdA. The responses of the suc and Ipd promoters to different environmental conditions and to regulator defects were investigated with appropriate IacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with IpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by s38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the Ipd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or s38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the Ipd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc suc Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.

The gerC region of Bacillus subtilis comprises a tricistronic operon, encoding enzymes that catalyse the late stages of menaquinone biosynthesis. The gerC58 mutation is responsible for a severe growth defect; unsuppressed mutant cells grow as very short rods, which sometimes septate aberrantly. Cultures grow only to a low cell density, rapidly lose viability, and never sporulate. Unlinked suppressor mutations can restore near-normal growth. Several independent suppressed isolates were examined; all grew to normal cell length, but they showed, to varying extents, a residual defect in the placement of the cell division septum. The germination properties of the suppressed derivatives varied from normal to significantly slow in germination in all germinants; therefore, the combination of the gerC mutation and different suppressor alleles resulted in spores with very different germination properties. This suggests that any relationship between the gerC gene products and spore germination is indirect. The gerCC58 mutation maps in a gene encoding the catalytic subunit of the heptaprenyl-diphosphate synthase, which is responsible for formation of the isoprenoid side chain of menaquinone-7, and it is proposed that the gerCA, gerCB and gerCC genes be renamed hepA, menG and hepB, respectively.

It was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.

A -lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF19 and its N-terminal amino acid sequence was determined. A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II. The bliB gene was isolated and sequenced. Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence. bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but -lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the me/C1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.

Bacteriophage M1 from the ruminal bacterium Selenomonas ruminantium strain ML12 comprises a 30 nm icosahedral capsid, a 25 nm tail and 48 kb of linear dsDNA with cohesive ends. A restriction map of the phage genome has been constructed. The presence of bacteriophage M1 in the rumen has been demonstrated by PCR amplification and Southern blot analysis of DNA from rumen bacterial samples obtained from ten different sheep. Lysogeny was demonstrated by hybridization of M1 DNA to host chromosomal DNA and by identification and cloning of a 2.3 kb region of the phage containing the predicted attP domain which promotes chromosomal integration. DNA sequencing of the attP region demonstrated two major ORFs surrounding the predicted attP site and structural analysis of this region revealed a motif comprising three different inverted repeats surrounding a 12 bp palindrome. Analysis of the translated amino acid sequence upstream of the attP site demonstrated the presence of conserved residues found within integrase proteins of several temperate phages of different bacterial species.

Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40.10. The eight early transcripts, all mapping in a 13 kb region adjacent to the attachment site of TP901-1, were present at maximal levels 10 min after infection. The four middle transcripts, observed at maximal levels 30 min after infection, are all located within a 2 kb region at the distal end of the early transcripts. The late class of transcripts were detected 40 min after infection and the amounts of these transcripts increased with time. The late transcripts were localized to the 13 kb region adjacent to the 2 kb middle transcribed region. The sequence of almost 4 kb of the early region was determined, allowing a detailed transcriptional map for the early region of which in total 6.4 kb was sequenced. Sequence analysis of the early region revealed two closely positioned but divergently orientated promoters, PL and PR, in accordance with the orientation of the ORFs and the transcriptional map. Nine ORFs were found, and similarities to a phage repressor, a single-stranded DNA-binding protein, a topoisomerase, a Cro-like protein and two other phage proteins of unknown function were detected. The gene arrangement in the early transcribed region of TP901-1 thus consists of two transcriptional units: one from PR containing four genes, of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter transcriptional unit.