- Volume 152, Issue 8, 2006

In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division- and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL–ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.

Pseudomonas aeruginosa PAO1 has two possible catabolic pathways of spermidine and spermine; one includes the spuA and spuB products with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH in P. aeruginosa PAO1 were characterized and the corresponding spdH gene in this strain identified. The deduced SpdH (620 residues, calculated M r of 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units (mg protein)−1 and a K m value of 36 μM. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units (mg protein)−1 and a K m of 18 μM. Knockout of spdH had no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, when spdH was fused to the polyamine-inducible promoter of spuA, it fully restored the ability of a spuA mutant to utilize spermidine. It is concluded that SpdH can perform a catabolic role in vivo, but P. aeruginosa PAO1 does not produce sufficient amounts of the enzyme to execute this function.

The reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) and other tetrazolium salts is widely used as an assay for bacterial, fungal and mammalian cell viability, but the genes encoding the reductase activities have not been defined. Here, it was shown that XTT and plasma membrane ferric reductase activities were 10–40-fold greater in Candida albicans than in Saccharomyces cerevisiae. XTT reductase activity was induced fivefold in C. albicans grown in low-iron conditions compared with iron-replete conditions, and for cells grown in unbuffered (pH 4.0–4.4) medium, XTT reductase activity was largely dependent on CaFRE10. XTT reductase activity of C. albicans grown in medium buffered to pH 6.8 was independent of CaFRE10 but, nonetheless, was upregulated in cells deprived of iron. Reduction of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), a membrane-permeable tetrazolium salt, occurred at an intracellular location and was independent of CaFRE10. However, MTT activity was induced by iron deprivation in C. albicans but not in S. cerevisiae. C. albicans possessed multiple iron- and pH-regulated reductase activities capable of reducing tetrazolium salts, but, when grown in unbuffered medium, CaFRE10 was required for XTT reductase activity.

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum are described. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. ∼90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankyrin repeat sequence (∼170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.

The toxin–antitoxin operon of pSM19035 encodes three proteins: the ω global regulator, the ε labile antitoxin and the stable ζ toxin. Accumulation of ζ toxin free of ε antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a ζ variant (ζY83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free ζ toxin induced stasis, but this was fully reversed by expression of the ε antitoxin within a defined time window. The time window for reversion of ζ toxicity by expression of ε antitoxin was dependent on the initial cellular level of ζ. After 240 min of constitutive expression, or inducible expression of high levels of ζ toxin for 30 min, expression of ε failed to reverse the toxic effect exerted by ζ in cells growing in minimal medium. Under the latter conditions, ζ inhibited replication, transcription and translation and finally induced death in a fraction (∼50 %) of the cell population. These results support the view that ζ interacts with its specific target and reversibly inhibits cell proliferation, but accumulation of ζ might lead to cell death due to pleiotropic effects.

Proteinase–adhesin complexes of Porphyromonas gingivalis wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpAcat, the Lys-specific proteinase Kgpcat and adhesin domains RgpAA1, RgpAA2, RgpAA3, KgpA1 and KgpA2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpAA4, indicated that this band contained C-terminally truncated KgpA1 (which has an identical N-terminus to RgpAA4) as well as the HagAA1* adhesin. Analysis of the Triton X-114 extracted complexes from the P. gingivalis isogenic mutants kgp (RgpA complex) and rgpA (Kgp complex) suggested that the Kgp complex consisted of Kgpcat, KgpA1 and KgpA2/HagAA2 and that the RgpA complex consisted of RgpAcat, RgpAA1, HagAA1*, RgpAA2 and RgpAA3. Each of the complexes was found to have equilibrium binding constants (K D) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower K D values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.

Streptococcus mutans is known as a primary pathogen responsible for dental caries. One of the virulence factors of S. mutans in cariogenicity is its ability to attach to the tooth surface and form a biofilm. Several surface proteins have been shown to be involved in this process. A 29 kDa surface protein named wall-associated protein A (WapA, antigen A or antigen III), was previously used as a vaccine in animal studies for immunization against dental caries. However, the function of WapA in S. mutans is still not clear. This study characterized the function of WapA in cell surface structure and biofilm formation. Compared to the wild-type, the wapA mutant had much-reduced cell chain length, diminished cell–cell aggregation, altered cell surface ultrastructure, and unstructured biofilm architecture. Furthermore, in vivo force spectroscopy revealed that the cell surface of the wapA mutant was less sticky than that of the wild-type cells. More interestingly, these phenotypic differences diminished as sucrose concentration in the medium was increased to 0.5 %. Real-time RT-PCR analysis demonstrated that sucrose strongly repressed wapA gene expression in both planktonic and biofilm cells. These results suggest that the WapA protein plays an important structural role on the cell surface, which ultimately affects sucrose-independent cell–cell aggregation and biofilm architecture.

Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.

The physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1.1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle, the glyoxylate shunt and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme, involved in the production of pyruvate, and pyruvate dehydrogenase, required for the channelling of pyruvate into acetyl-CoA, were also induced in IAA-treated cells. Moreover, it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression, enzyme activity and metabolite level involved in central metabolic pathways in E. coli.

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1H-NMR and mass spectroscopy and their antibacterial activity was assayed.

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.

Using a yeast two-hybrid assay system, it was demonstrated that the four-helix bundle of the Rhodobacter sphaeroides PrrB histidine kinase both serves as the interaction site for the regulatory domain of its cognate response regulator PrrA and is the primary determinant of the interaction specificity. The α-helix 1 and its flanking turn region within the dimerization domain (DD) of the PrrB histidine kinase appear to play an important role in conferring the recognition specificity for the PrrA response regulator on the DD. The catalytic ATP-binding domain of the histidine kinase, which functions as the catalytic unit for the phosphotransfer reaction from ATP to the conserved histidine residue in the DD, also appears to contribute to the enhancement of the recognition specificity conferred by the DD. It was also revealed that replacement of Asp-63 and Lys-113 of the PrrA response regulator by alanine abolished protein–protein interactions between PrrA and its cognate histidine kinase PrrB, whereas mutations of Asp-19, Asp-20 and Thr-87 to alanine did not affect protein–protein interactions, indicating that among the active site residues of PrrA, Asp-63 and Lys-113 are important not only in the function of PrrA but also for protein–protein interactions between PrrA and PrrB.

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16.4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13.5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13.5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N11-A motif) for ArhR was found upstream from the transcription start point of arhA3.

Toluene and other fuel hydrocarbons are commonly found in association with radionuclides at numerous US Department of Energy sites, frequently occurring together with Cr(VI) and other heavy metals. In this study, the extremely radiation-resistant bacterium Deinococcus radiodurans, which naturally reduces Cr(VI) to the less mobile and less toxic Cr(III), was engineered for complete toluene degradation by cloned expression of tod and xyl genes of Pseudomonas putida. The recombinant Tod/Xyl strain showed incorporation of carbon from 14C-labelled toluene into cellular macromolecules and carbon dioxide, in the absence or presence of chronic ionizing radiation. The engineered bacteria were able to oxidize toluene under both minimal and complex nutrient conditions, and recombinant cells reduced Cr(VI) in sediment microcosms. As such, the Tod/Xyl strain could provide a model for examining the reduction of metals coupled to organic contaminant oxidation in aerobic radionuclide-contaminated sediments.

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.