- Volume 153, Issue 3, 2007

In vitro experiments have shown that the genes of the de novo pyrimidine biosynthetic pathway of Bacillus subtilis, the pyr genes, are regulated by a transcriptional attenuation mechanism. Specific regulatory sequences (binding loops, BLs) are located within three untranslated leader sequences at the beginning of pyr mRNA. These binding loops, BL1, BL2 and BL3, act as anti-antiterminators of transcription when stabilized by the regulator protein PyrR. In this work, the interaction of PyrR with BL1, BL2 and BL3 was qualitatively and quantitatively analysed in vivo using the yeast three-hybrid system. The results indicate that PyrR specifically binds to BL1, BL2 and BL3. Furthermore, the data suggest that the strength of interaction between PyrR and the three different BLs in vivo is within the same dimension. The yeast three-hybrid system also proved to be useful for the rapid analysis of structural requirements for PyrR–BL binding. Point mutations within the predicted critical regions of BL1, BL2 and BL3 led to drastically reduced binding of PyrR. In summary, it is shown that the yeast three-hybrid system is well suited to qualitatively and quantitatively analyse bacterial regulatory systems that are based on factor-independent transcriptional attenuation.

Transcription of the Bacillus subtilis kdgRKAT operon, which comprises genes involved in the late stage of galacturonate utilization, is known to be negatively regulated by the KdgR repressor. In this study, Northern analysis was carried out to demonstrate that the kdgR gene also negatively regulates the kduID operon, encoding ketodeoxyuronate isomerase and ketodeoxygluconate reductase. It has also been demonstrated that expression of the kduID operon can be induced by galacturonate and is subject to catabolite repression by glucose. The ccpA gene was found to be involved in this catabolite repression. Primer extension analysis identified a σ A-like promoter sequence preceding kduI. Gel mobility shift assays and DNase I footprinting analyses indicated that KdgR is capable of binding specifically to two sites within the kdgR–kduI intergenic region in vitro. Reporter gene analysis revealed that these two KdgR-binding sites function in vivo. One site is centred 33.5 bp upstream of the translational start site of kdgR and can serve as an operator for controlling expression of the kdgRKAT operon. The other is centred 57.5 bp upstream of the translational start site of kduI and can serve as an operator for controlling expression of the kduID operon. Possible physiological significance of this regulation is discussed.

The Synechococcus sp. PCC 7942 nitrogen regulator PipX interacts in a 2-oxoglutarate-dependent manner with the global nitrogen transcription factor NtcA and the signal transduction protein PII. In vivo, PipX is involved in the NtcA-dependent induction of glnB and glnN genes. To further investigate the extent to which PipX is involved in global nitrogen control, the effect of pipX inactivation on various nitrogen-regulated processes was determined. The PipX-deficient mutant was able to use nitrate as a nitrogen source and to efficiently inhibit the nitrate transport upon ammonium addition but showed decreased nitrate and nitrite reductase activities and a delay in the induction of nitrate utilization after transfer of cultures from ammonium- to nitrate-containing media. In contrast to the wild-type, glutamine synthetase activity was not upregulated upon depletion of combined nitrogen from cultures of the mutant strain. Inactivation of pipX impaired induction of nblA and delayed phycobilisome degradation, but did not affect recovery of nitrogen-deprived cultures. Taken together, the results indicate that PipX interacts with NtcA to facilitate efficient acclimation of cyanobacteria to conditions of nitrogen limitation.

Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. β-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase- and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine- and 3-phosphohydroxyasparagine-containing CDAs, the ΔhasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, ΔasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the β-hydroxylation and phosphorylation of the Asn residue within CDA.

Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12 bp RNA–DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35 bp DNA–RNA–DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should cntribute to the analysis of RNase Hs of other parasite species.

For osmoprotection, Escherichia coli can synthesize glycine betaine from externally supplied choline by the Bet system (betTIBA products). The major carrier of choline is the high-affinity, proton-driven, secondary transporter BetT, which belongs to the BCCT family of transporters. Fusion proteins consisting of N-terminal fragments of BetT linked to β-galactosidase (LacZ) or alkaline phosphatase (PhoA) were constructed. By analysis of 51 fusion proteins with 37 unique fusion-points, the predictions that BetT comprised 12 membrane-spanning regions and that its N- and C-terminal extensions of about 12 and 180 amino acid residues, respectively, were situated in the cytoplasm were confirmed. This is believed to represent the first experimental examination of the membrane topology of a BCCT family protein. Osmotic upshock experiments were performed with spectinomycin-treated E. coli cells that had expressed the wild-type or a mutant BetT protein during growth at low osmolality (160 mosmol kg−1). The choline transport activity of wild-type BetT increased tenfold when the cells were stressed with 0.4 M NaCl (total osmolality 780 mosmol kg−1). The peak activity was recorded 5 min after the upshock and higher or lower concentrations of NaCl reduced the activity. Deletions of 1–12 C-terminal residues of BetT caused a gradual reduction in the degree of osmotic activation from ten- to twofold. Mutant proteins with deletion of 18–101 residues displayed a background transport activity, but they could not be osmotically activated. The data showed that the cytoplasmic C-terminal domain of BetT plays an important role in the regulation of BetT activity and that C-terminal truncations can cause BetT to be permanently locked in a low-transport-activity mode.

tRNA-encoding genes (tDNA) are known hot-spots for the integration of ecto-chromosomal DNA (ECDNA) including genomic islands. However, only a few loci are currently known to be targeted by such insertions in Escherichia coli. A PCR-based screening of tDNA integrity was therefore performed on a collection of E. coli strains in order to identify tDNA loci that are most frequently intact and those that are preferred ECDNA insertion sites. It was shown that only a subset of tDNAs were hot-spots for ECDNA insertions, and that the majority of loci were never targeted by such insertions. Polycistronic tDNAs, highly transcribed tDNAs or tDNAs encoding tRNAs recognizing frequently used codons were generally not targeted by ECDNA insertions. Most interestingly, strains of different ECOR groups showed different patterns of tDNA loci polymorphism. More subtle differences were also observed between strains of different pathotypes.

The fission yeast Schizosaccharomyces pombe is widely used as a model eukaryote for cell and molecular studies but little is known of natural genetic variation in this species. In order to obtain informative molecular markers, imperfect tandem repeats, identified through bioinformatic methods, were tested for length polymorphism in six wild-type strains of Sch. pombe isolated from different substrates and geographical locations in Africa, America, Asia and Europe. Of 26 loci tested, 21 were multi-allelic, consistent with tandem repeat copy number variation. Eleven of these polymorphic tandem repeats are in regions encoding intracellular proteins. Most of the protein-coding repeats are not sited within structured domains but have non-regular predicted structure; one has a repeat unit length corresponding to integer turns of a predicted amphipathic α-helix secondary structure, suggesting that this repeat may be tolerated because copy number mutations change α-helix length but not orientation within the protein structure. In contrast to the differences observed between natural isolates of Sch. pombe, genetic strains were found to be essentially isogenic: only two polymorphic loci were detected out of 26 minisatellites and five microsatellites tested in 16 strains, including a hypervariable microsatellite in the med15 gene. The polymorphic tandem repeat markers identified in this study will prove useful for DNA fingerprinting and molecular analysis of natural genetic variation in Sch. pombe isolates.

Phytophthora infestans is the cause of late blight, a devastating and re-emerging disease of potato. Significant advances have been made in understanding the biology of P. infestans, and in the development of molecular tools to study this oomycete. Nevertheless, little is known about the molecular bases of the establishment or development of disease in this hemibiotrophic pathogen. Suppression subtractive hybridization (SSH) was used to generate cDNA enriched for sequences upregulated during potato infection. To identify pathogen-derived cDNAs, and eliminate host sequences from further study, SSH cDNA was hybridized to a P. infestans bacterial artificial chromosome library. A new gene family was identified called Pinci1, comprising more than 400 members arranged in clusters of up to nine copies throughout the P. infestans draft genome sequence. Real-time RT-PCR was used to quantify the expression of five classes of transcript within the family, relative to the constitutively expressed PiactA gene, and it revealed them to be significantly upregulated from 12 to 33 h post-inoculation, a period defining the biotrophic phase of infection. Computational analysis of sequences suggested that transcripts were non-protein coding, and this was confirmed by transient expression of FLAG-tagged ORFs in P. infestans.

Recent studies with Myxococcus xanthus have suggested that homologues of the Escherichia coli heat-shock sigma factor, RpoH, may not be involved in the heat-shock response in this δ-proteobacterium. The genome of another δ-proteobacterium, Geobacter sulfurreducens, which is considered to be a representative of the Fe(III)-reducing Geobacteraceae that predominate in a diversity of subsurface environments, contains an rpoH homologue. Characterization of the G. sulfurreducens rpoH homologue revealed that it was induced by a temperature shift from 30 °C to 42 °C and that an rpoH-deficient mutant was unable to grow at 42 °C. The predicted heat-shock genes, hrcA, grpE, dnaK, groES and htpG, were heat-shock inducible in an rpoH-dependent manner, and comparison of promoter regions of these genes identified the consensus sequences for the −10 and −35 promoter elements. In addition, DNA elements identical to the CIRCE consensus sequence were found in promoters of rpoH, hrcA and groES, suggesting that these genes are regulated by a homologue of the repressor HrcA, which is known to bind the CIRCE element. These results suggest that the G. sulfurreducens RpoH homologue is the heat-shock sigma factor and that heat-shock response in G. sulfurreducens is regulated positively by RpoH as well as negatively by the HrcA/CIRCE system.

Genome-wide data mining indicated that six genes (ncgl1031, ncgl2302, ncgl2325, ncgl2326, ncgl2922 and ncgl2953) encoding putative transport proteins are involved in uptake of various aromatic compounds that are further degraded through the β-ketoadipate, gentisate and resorcinol pathways in Corynebacterium glutamicum. The gentisate (GenK/NCgl2922) and vanillate (VanK/NCgl2302) transporters have been identified previously. In this study, physiological functions of the remaining four putative transporters as well as the vanillate transporter (VanK/NCgl2302) were examined by genetic disruption/complementation and uptake assays. Results indicated that ncgl1031 encodes PcaK for 4-hydroxybenzoate and protocatechuate transport, and ncgl2302 encodes VanK for vanillate transport. Genetic studies and uptake assays indicated that both ncgl2325/benK and ncgl2326/benE are involved in benzoate transport in C. glutamicum. When growth rates were compared for two benzoate transporter mutants, benK and benE, a high growth rate was observed for the benE mutant. Sequence alignments revealed that PcaK, VanK, BenK and GenK belong to the major facilitator superfamily (MFS). Modelling of secondary structures based on previously characterized MFS members revealed that NCgl1031, NCgl2302, NCgl2325 and NCgl2922 are typical 12 helix transmembrane proteins but NCgl2326 contains only 11 α-helices. Thus the functionally identified NCgl2326 belongs to a novel type of benzoate transporters. Attempts to identify the phenotype of a hydK/ncgl2953 mutant failed, so the function of ncgl2953 remains unclear.

The hypothetical protein Cpn1027 was detected in the inclusion membrane of Chlamydia pneumoniae-infected cells with antibodies raised with Cpn1027 fusion proteins in an indirect immunofluorescence assay. The inclusion membrane staining by the anti-Cpn1027 antibodies co-localized with the staining of an antibody recognizing a known inclusion membrane protein designated IncA and these membrane stainings were blocked by the corresponding but not irrelevant fusion proteins. Although Cpn1027 was not predicted to be an inclusion membrane protein, it contained a bi-lobed hydrophobic domain region at its N-terminus, a signature secondary structural motif possessed by most chlamydial inclusion membrane proteins. The Cpn1027 protein was detected as early as 12 h after C. pneumoniae infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of Cpn1027 via a transgene failed to affect the subsequent chlamydial infection. The anti-Cpn1027 polyclonal antisera failed to detect any significant signals in cells infected with chlamydial species other than C. pneumoniae, which is consistent with the sequence analysis result that no significant homologues of Cpn1027 were found in any other species. These experiments together have demonstrated that Cpn1027 is a newly identified inclusion membrane protein unique to C. pneumoniae.