- Volume 153, Issue 9, 2007

A virulent phage (φCR1) capable of generalized transduction in Citrobacter rodentium was isolated from the environment and characterized. C. rodentium is a natural pathogen of mice, causing transmissible murine colonic hyperplasia. Sequencing of its genome has recently been completed and will soon be fully annotated and published. C. rodentium is an important model organism for infections caused by the human pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC). φCR1 uses a lipopolysaccharide receptor, has a genome size of approximately 300 kb, and is able to transduce a variety of markers. φCR1 is the first reported transducing phage for C. rodentium and will be a useful tool for functional genomic analysis of this important natural murine pathogen.

Intracellular copper homeostasis in bacteria is maintained as the result of a complex ensemble of cellular processes that in Escherichia coli involve the coordinated action of two systems, cue and cus. In contrast, the pathogenic bacterium Salmonella harbours only the cue regulon, including copA, which is shown here to be transcriptionally controlled by CueR. Mutant strains in the CueR-regulated genes were constructed to characterize the response of Salmonella enterica serovar Typhimurium to high concentrations of extracellular copper under both aerobic and anaerobic conditions. Unlike its counterpart in E. coli, inactivation of cuiD displays the most severe phenotype and is also required for copper tolerance under anaerobic conditions. Deletion of copA has a mild effect in aerobiosis, but strongly impairs survival in the absence of oxygen. In a ΔcopA strain, a second Salmonella-specific P-type ATPase, GolT, can substitute the copper transporter, diminishing the effect of its deletion. The overall results highlight the importance of the cue system for controlling intracellular copper stress. The observed differences between Salmonella and E. coli in handling copper excess may contribute to our understanding of the distinct capability of these related pathogenic bacteria to survive outside the host.

SipB (593 aa), one of the Salmonella invasion proteins (Sips), is secreted via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Here, we report the delineation of several functional regions present in the SipB protein. Our data show that residues 3–8 of the SipB protein are essential for its secretion from the bacterial cell and that the SicA chaperone, which is important to ensure stability of SipB and SipC in the bacterial cytosol, binds to SipB somewhere between amino acids 80 and100 of the SipB N-terminal region. Interestingly, the N-terminal region (residues 1–160) of SipB (SipB160) cannot be secreted via the SPI-1 T3SS, but fusion of the C-terminal amphipathic region (residues 300–593) to SipB160 can restore secretion via this system.

Comparative genomics reveals a common theme of 20S proteasome and proteasome-activating nucleotidase genes dispersed throughout archaeal genomes yet arranged in conserved linkages with gene homologues of translation and/or transcription machineries. To provide biological evidence for these linkages as well as insight into proteasome operon organization, transcripts of the five proteasomal genes of the halophilic archaeon Haloferax volcanii were analysed by Northern (RNA) blotting, RT-PCR and primer extension. These included psmA, psmB and psmC, encoding the 20S proteasomal subunits α1, β and α2, as well as panA and panB, encoding the PanA and PanB proteasome-activating nucleotidase proteins, respectively. All five of these genes are dispersed throughout the H. volcanii genome. For each proteasomal gene, a distinct transcript was detected by Northern blotting that was similar in size to the respective coding region. For both psmA and psmC, an additional transcript was detected that was 1.34 and 0.85 kb greater, respectively, than the coding region. Further analysis by Northern blotting and RT-PCR revealed that psmA was co-transcribed with genes encoding a Pop5 homologue of the RNase P endoRNase as well as an S-adenosylmethionine (SAM)-dependent methyltransferase. Likewise, psmC was co-transcribed with a downstream gene encoding a molybdenum cofactor sulfurase C-terminal (MOSC) domain protein. Additional proteasomal and neighbouring gene-specific transcriptional linkages were detected by RT-PCR. These results provide the first evidence that proteasome and tRNA modification genes are co-transcribed, reveal that a number of additional enzymes including those predicted to facilitate metal–sulfur cluster assembly are co-regulated with proteasomes at the transcriptional level, and provide further insight into proteasome gene transcription in archaea.

White-rot fungi secret a large number of hydrolytic and oxidative enzymes for degradation of lignocellulosic material. The sequencing of the genome of the white-rot fungus Phanerochaete chrysosporium has facilitated the characterization of its complete extracellular proteome. P. chrysosporium was grown on liquid medium, containing glucose, cellulose or wood chips as the carbon source, and also in solid substrate fermentation bags. For liquid-grown cultures, the extracellular protein fraction was separated by 2D gel electrophoresis. Protein spots were analysed by in-gel digestion and liquid chromatography (LC)/MS/MS. A total of 18 additional protein spots from the 2D gels yielded hits from blast searches. From solid substrate cultures in which the fungus was grown in bags, the proteins were resolved by SDS-PAGE, subjected to in-gel digestion and then identified by LC/MS/MS. An additional 16 proteins yielded hits on blast searches. Enzymes involved in cellulose, hemicellulose, lignin and protein degradation were identified. Expression patterns were very similar between cellulose-grown cultures and wood-grown cultures. In addition to enzymes which act on lignocellulosic material, proteases were also found, indicating the need of fungi to scavenge for nitrogen in wood.

Two genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K+ transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K+-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Δ trk2Δ mutant of Saccharomyces cerevisiae, unable to grow at low K+. This expression resulted in partial recovery of growth and ability to retain K+ at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb+ uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K+ transporters exhibiting a sigmoid response against Rb+ concentration and presenting a deviation from classic Michaelis–Menten kinetics at low substrate concentrations. Rb+ uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na+ at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD+-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k cat/K m with NADH)/(k cat/K m with NADPH)] compared with the wild-type (WT), which was due to decrease of k cat with NADPH in the R276H mutant and increase of K m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP+ and NADH/NAD+ ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

The degradation of taurine, isethionate and sulfoacetate in Cupriavidus necator (Ralstonia eutropha) H16 was shown by enzyme assays to be inducible, and each pathway involved sulfoacetaldehyde, which was subject to phosphatolysis by a common sulfoacetaldehyde acetyltransferase (Xsc, H16_B1870) to yield acetyl phosphate and sulfite. The neighbouring genes encoded phosphate acetyltransferase (Pta, H16_B1871) and a hypothetical protein [domain of unknown function (DUF)81, H16_B1872], with eight derived transmembrane helices. RT-PCR showed inducible transcription of these three genes, and led to the hypothesis that H16_B1872 and orthologous proteins represent a sulfite exporter, which was named TauE.

Chromomycin A3 is an antitumour antibiotic that acts by inhibiting transcription and replication of DNA. The producer micro-organism Streptomyces griseus subsp. griseus is highly resistant to chromomycin A3 and to the structurally related compound mithramycin upon induction with chromomycin A3. The biosynthetic gene cluster of chromomycin contains three genes involved in self-resistance to chromomycin in S. griseus: cmrA and cmrB encode a type I ATP-binding cassette (ABC) transporter, and cmrX encodes a UvrA-like protein of ABC excision nuclease systems. These genes are linked in the chromosome, together with a gene encoding a transcriptional repressor (cmmRII). Involvement of these genes in chromomycin resistance was determined through gene inactivation, and heterologous expression in Streptomyces albus. Inactivation of cmrX produced a chromomycin-sensitive low-producer strain, while inactivation of cmmRII generated a high-chromomycin-producer strain, which was resistant to chromomycin, and also to mithramycin. Expression of either cmrA and cmrB, or cmrX, in S. albus generated strains with low chromomycin resistance; it was therefore necessary to co-express the three genes to achieve high levels of resistance. However, the CmrAB ABC transporter conferred a high level of resistance to the biosynthesis intermediate 4A,4E-O-dideacetyl-chromomycin A3. A model is proposed for the biosynthesis of, and self-resistance to, chromomycin A3 in S. griseus subsp. griseus.

Novel plasmids were constructed for the analysis of DNA fragments from the rumen bacterium Pseudobutyrivibrio ruminis. Five previously unidentified promoters were characterized using a novel primer extension method to identify transcription start sites. The genes downstream of these promoters were not identified, and their activity in expression of genomic traits in wild-type P. ruminis remains putative. Comparison with promoters from this and closely related species revealed a consensus sequence resembling the binding motif for the RNA polymerase σ 70-like factor complex. Consensus −35 and −10 sequences within these elements were TTGACA and ATAATATA respectively, interspaced by 15–16 bp. The consensus for the −10 element was extended by one nucleotide upstream and downstream of the standard hexamer (indicated in bold). Promoter strengths were measured by reverse transcription quantitative PCR and β-glucuronidase assays. No correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. However, a mutation within the −35 element of one promoter revealed that transcriptional strength and choice of transcription start site were sensitive to this single nucleotide change.

A temperate transposable bacteriophage (MP22) was isolated from a Korean clinical isolate of Pseudomonas aeruginosa. It has a coliphage λ-like morphology and a double-stranded DNA genome. The complete nucleotide sequence and annotation of the MP22 genome and its characteristics are presented. The MP22 genome is 36 409 bp long with a G+C content of 64.2 mol%. The genome contains 51 proposed ORFs, of which 48 (94 %) display synteny and significant nucleotide and protein sequence similarity to the corresponding ORFs of the closely related phage, D3112. Three of the predicted ORFs are unique proteins, whose functions are yet to be revealed. The phage c repressors exhibit striking dissimilarities and, when present as a single gene, did not show cross-immunity. In contrast, although an MP22 lysogen could be productively infected with D3112, MP22 could not grow on a D3112 lysogen, indicating a role of other D3112 genes in superinfection exclusion.

Fis is a nucleoid-associated protein in Escherichia coli that is abundant during early exponential growth in rich medium but is in short supply during stationary phase. Its role as a transcriptional regulator has been demonstrated for an increasing number of genes. In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium, DNA microarray analyses were conducted in fis and wild-type strains during early, mid-, late-exponential and stationary growth phases. The results uncovered 231 significantly regulated genes that were distributed over 15 functional categories. Regulatory effects were observed at all growth stages examined. Coordinate upregulation was observed for a number of genes involved in translation, flagellar biosynthesis and motility, nutrient transport, carbon compound metabolism, and energy metabolism at different growth stages. Coordinate down-regulation was also observed for genes involved in stress response, amino acid and nucleotide biosynthesis, energy and intermediary metabolism, and nutrient transport. As cells transitioned from the early to the late-exponential growth phase, different functional categories of genes were regulated, and a gradual shift occurred towards mostly down-regulation. The results demonstrate that the growth phase-dependent Fis expression triggers coordinate regulation of 15 categories of functionally related genes during specific stages of growth of an E. coli culture.

Nitrogen assimilation is important during solvent production by Clostridium saccharobutylicum NCP262, as acetone and butanol yields are significantly affected by the nitrogen source supplied. Growth of this bacterium was dependent on the concentration of organic nitrogen supplied and the expression of the assimilatory enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT), was shown to be induced in nitrogen-limiting conditions. The regions flanking the gene encoding GS, glnA, were isolated from C. saccharobutylicum genomic DNA, and DNA sequencing revealed that the structural genes encoding the GS (glnA) and GOGAT (gltA and gltB) enzymes were clustered together with the nitR gene in the order glnA-nitR-gltAB. RNA analysis showed that the glnA-nitR and the gltAB genes were co-transcribed on 2.3 and 6.2 kb RNA transcripts respectively, and that all four genes were induced under the same nitrogen-limiting conditions. Complementation of an Escherichia coli gltD mutant, lacking a GOGAT small subunit, was achieved only when both the C. saccharobutylicum gltA and gltB genes were expressed together under anaerobic conditions. This is believed to be the first functional analysis of a gene cluster encoding the key enzymes of nitrogen assimilation, GS and GOGAT. A similar gene arrangement is seen in Clostridium beijerinckii NCIMB 8052, and based on the common regulatory features of the promoter regions upstream of the glnA operons in both species, we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as antisense RNA.

The σ 54-dependent transcriptional regulator SfnR is essential for the use of dimethyl sulfone (DMSO2) as a sulfur source by Pseudomonas putida DS1. SfnR binds three SfnR-binding sites (sites 1, 2 and 3) within an intergenic region of the divergently transcribed sfnAB and sfnFG gene clusters. The site 1 region, proximal to the sfnF gene, is indispensable for the expression of the sfnFG operon, which encodes components of DMSO2 monooxygenase. We investigated the transcriptional regulation of the sfnAB operon and possible functions of the sfnA gene. RT-PCR analysis revealed that the sfnAB gene cluster, which is similar to homologues of the acyl-CoA dehydrogenase family, was transcribed as an operon, and its expression was regulated by SfnR under conditions of sulfate starvation. Deletion analyses using lacZ as a reporter demonstrated that the region up to at least −138 bp from the transcription start point of sfnA (containing sites 2 and 3) was necessary for the expression of the sfnAB operon. A growth test of the sfnA-disrupted mutant revealed the possibility that sfnA may be involved in the use of methanethiol as a sulfur source.

A previously unannotated, putative fliK gene was identified in the Campylobacter jejuni genome based on sequence analysis; deletion mutants in this gene had a ‘polyhook’ phenotype characteristic of fliK mutants in other genera. The mutants greatly overexpressed the σ 54-dependent flagellar hook protein FlgE, to form unusual filamentous structures resembling straight flagella in addition to polyhooks. The genome sequence reveals only one gene predicted to encode an orthologue of the NtrC-family activator required for σ 54-dependent transcription. Hence, all σ 54-dependent genes in the genome would be overexpressed in the fliK mutant together with flgE. Microarray analysis of genome-wide transcription in the mutant showed increased transcription of a subset of genes, often downstream of σ 54-dependent promoters identified by a quality-predictive algorithm applied to the whole genome. Assessment of genome-wide transcription in deletion mutants in rpoN, encoding σ 54, and in the σ 54-activator gene flgR, showed reciprocally reduced transcription of genes that were overexpressed in the fliK mutant. The fliA (σ 28)-dependent regulon was also analysed. Together the data clearly define the roles of the alternative sigma factors RpoN and FliA in flagellar biogenesis in C. jejuni, and identify additional putative members of their respective regulons.