- Volume 154, Issue 2, 2008

Microbial genome sequencing has, for the first time, made accessible all the components needed for both the elaboration and the functioning of a cell. Associated with other global methods such as protein and mRNA profiling, genomics has considerably extended our knowledge of physiological processes and their diversity not only in human, animal and plant pathogens but also in environmental isolates. At a higher level of complexity, the so-called meta approaches have recently shown great promise in investigating microbial communities, including uncultured micro-organisms. Combined with classical methods of physico-chemistry and microbiology, these endeavours should provide us with an integrated view of how micro-organisms adapt to particular ecological niches and participate in the dynamics of ecosystems.

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTSGlcNAc), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc2) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in ΔwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the ΔwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

Serratia marcescens is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of sdeAB was identified (sdeR) and found to be 40 % homologous to MarA, an Escherichia coli transcriptional regulator. To provide conclusive evidence as to the role of these components in S. marcescens, sdeB, hasF and sdeR deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into S. marcescens UOC-67 (wild-type) and, for sdeB and hasF, T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The sdeB, hasF and sdeR knockout strains were consistently more susceptible to antibiotics than the parent strains, with the sdeB/hasF double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the sdeB or hasF genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the sdeB/hasF double knockout. Antibiotic accumulation assays for the sdeB knockout mutant strains performed in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the sdeB and the hasF individual knockouts, we conclude that S. marcescens HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for sdeR-deficient and overexpressing strains confirm that SdeR is an activator of sdeAB and acts to enhance the overall multidrug resistance of S. marcescens.

High hydrostatic pressure processing (HPP) is currently being used as a treatment for certain foods to control the presence of food-borne pathogens, such as Listeria monocytogenes. Genomic microarray analysis was performed to determine the effects of HPP on L. monocytogenes in order to understand how it responds to mechanical stress injury. Reverse transcriptase PCR analysis of tufA and rpoC indicated that the reduction of mRNA expression in HPP-treated cells was dependent on intensity and time of the treatment. Treatments of 400 and 600 MPa for 5 min on cells in the exponential growth phase, though leading to partial or complete cellular inactivation, still resulted in measurable relative differential gene expression. Gene set enrichment analysis indicated that HPP induced increased expression of genes associated with DNA repair mechanisms, transcription and translation protein complexes, the septal ring, the general protein translocase system, flagella assemblage and chemotaxis, and lipid and peptidoglycan biosynthetic pathways. On the other hand, HPP appears to suppress a wide range of energy production and conversion, carbohydrate metabolism and virulence-associated genes accompanied by strong suppression of the SigB and PrfA regulons. HPP also affected genes controlled by the pleotrophic regulator CodY. HPP-induced cellular damage appears to lead to increased expression of genes linked to sections of the cell previously shown in bacteria to be damaged or altered during HPP exposure and suppression of gene expression associated with cellular growth processes and virulence.

In Escherichia coli, osmoregulated periplasmic glucans (OPGs) are highly substituted by phosphoglycerol, phosphoethanolamine and succinyl residues. A two-step model was proposed to account for phosphoglycerol substitution: first, the membrane-bound phosphoglycerol transferase I transfers residues from membrane phosphatidylglycerol to nascent OPG molecules; second, the periplasmic phosphoglycerol transferase II swaps residues from one OPG molecule to another. Gene opgB was reported to encode phosphoglycerol transferase I. In this study, we demonstrate that the periplasmic enzyme II is a soluble form of the membrane-bound enzyme I. In addition, timing of OPG substitution was investigated. OPG substitution by succinyl residues occurs rapidly, probably during the backbone polymerization, whereas phosphoglycerol addition is a very progressive process. Thus, both phosphoglycerol transferase activities appear biologically necessary for complete OPG substitution.

In Mycobacterium tuberculosis, hspR is the last gene of the dnaKJE operon. It encodes the repressor HspR, which regulates the expression from this operon by binding to a consensus upstream sequence known as HAIR (HspR-associated inverted repeats). Previous investigations in the related Gram-positive bacterium Streptomyces coelicolor have revealed that DnaK acts as a co-repressor for HspR. In this investigation, a similar situation was encountered using the corresponding mycobacterial pair. However, the novel feature unearthed in this study is that the mycobacterial GroELs, GroEL1 and GroEL2, considerably stimulate the HAIR-binding activity of the HspR-DnaK combination. That these GroELs play a role in the folding process was evident from the observation that when heat- or chemically denatured HspR was renatured, the protein gained optimal activity only if one of these GroEL class chaperones was present along with DnaK. The renaturation process was found to be dependent on ATP hydrolysis. The DnaK-dependent DNA-binding activity of HspR could also be stimulated by DnaJ, but GrpE, which is known to release DnaK-bound substrates, was found to be inhibitory. The results of this study suggest that protein folding plays a substantial role in the activation of HspR following heat shock and that DnaK may be involved in two ways – first, as a chaperone acting in concert with GroEL and/or DnaJ and second, as a co-repressor bound to HspR.

We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.

In Escherichia coli K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is shown to be the first gene of a six-gene operon (ydhYVWXUT) that encodes proteins predicted to be components of an oxidoreductase. Mapping the ydhY–T transcript start and site-directed mutagenesis confirmed that the ydhY–T genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site is centred at −42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed ydhY–T expression. Analysis of the DNA sequence of the ydhY promoter region (PydhY) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at −42, −16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression, whereas mutation of heptamer −42 and heptamer −16 did not affect the response to nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the −16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL–PydhY complexes in gel retardation assays. Mutation of heptamer −42, which is located within the FNR binding site, or heptamer +6 (but not heptamers −16 or +15) impaired nitrate-mediated repression. Thus, although the region of the ydhY–T promoter containing the −16 and +15 heptamers was recognized by NarL in vitro, mutation of these heptamers did not affect NarL-mediated repression in vivo.

Typical enteropathogenic Escherichia coli (EPEC) O55 : H7 is regarded as the closest relative of enterohaemorrhagic E. coli (EHEC) O157 : H7. Both serotypes usually express the γ1 intimin subclass and trigger actin polymerization by the Tir-TccP pathway. However, atypical O55 : H7 strains capable of triggering actin polymerization via the Tir-Nck pathway have recently been identified. In this study, we investigated the genotypic differences and phylogenetic relationships between typical and atypical O55 : H7 strains. We show that the atypical O55 : H7 strains, which express the θ intimin subclass and lack both tccP and tccP2, belong to an E. coli lineage distinct from the typical O55 : H7 and from the EPEC O55 : H6, which also uses the Tir-Nck actin polymerization pathway. We conducted genomic comparisons of the chromosomal regions covering the O-antigen gene cluster and its flanking regions between the three O55 lineages by RFLP analysis of PCR products and DNA sequencing analysis of about 65 kb chromosomal regions. This unexpectedly revealed that horizontal transfer of large fragments (≥40 kb) encoding the O55-antigen gene cluster and part of the neighbouring colanic acid gene cluster was involved in the emergence of the three O55 E. coli lineages. The data provide new insights into the mechanisms involved in the generation of a wide variety of O-serotypes in Gram-negative bacteria.

Two-component systems (TCSs) are a large family of signalling pathways characterized by the successive transfer of phosphoryl groups between the histidine and aspartate residues of paired histidine kinase and response regulator proteins. With the availability of genome sequences for four genera of myxobacteria it has become possible to assess the genomic complements of myxobacterial TCS genes and to characterize features of their organization and evolutionary heritage. In this study we have compiled lists of the TCS genes within myxobacterial genomes and characterized their domain architecture, gene organization and evolutionary relationships. In order to provide an appropriate context for our conclusions, where possible we have compared myxobacterial TCSs with those found in 316 other completely sequenced bacteria. Myxobacteria have the largest number of TCSs of any organisms. An unusually low proportion of TCS genes are paired in myxobacterial genomes, and myxobacterial histidine kinases also seem to sense internal signals to an unusual degree. Phylogenetic evidence has allowed us to suggest homologous relationships of proteins across the myxobacteria, and it appears that myxobacterial TCS evolution has been dominated by duplications, gene rearrangements and changes in sensory domain complements. The systematic classification of the TCS proteins of the myxobacteria presented here should also provide a framework for future experimental studies on two-component regulation in these organisms.

A fusellovirus SSV4 and a pRN-like plasmid pXZ1 were co-isolated from a single strain of Sulfolobus. In contrast to the previously characterized virus–plasmid hybrids pSSVx and pSSVi, which can coexist intracellulary with a fusellovirus, pXZ1 is not packaged into viral particles and shows no viral infectivity. The virus and plasmid carry genomes of 15 135 and 6970 bp, respectively. For SSV4, 33 predicted ORFs are compactly organized with a strong preference for UGA stop codons, three-quarters of which overlap with either the Shine–Dalgarno motif or the start codon of the following gene. pXZ1 carries seven ORFs, three of which encode an atypical RepA, a PlrA and a CopG protein. A fourth ORF exhibits a high nucleotide sequence identity to the SSV4 integrase gene, which suggests that it has been transferred to the plasmid from SSV4. A single point mutation within an otherwise identical 500 bp region of the integrase gene occurs in the viral attachment site (attP), which corresponds to the anticodon region of the targeted tRNA gene in the host chromosome. This point mutation confers on pXZ1 the ability to integrate into the tRNAGlu[CUC] gene, which differs from the integration site of SSV4, tRNAGlu[UUC]. SSV4 and pXZ1 were also shown experimentally to integrate into separate sites on the host chromosome. This is believed to be the first report of a pRN plasmid sharing its natural host with a fusellovirus and carrying a highly similar integrase gene.

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CuSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

Pseudomonas aeruginosa PtxR enhances the expression of the exotoxin A gene toxA. The expression of ptxR itself, which occurs from two promoters (P1 and P2), is not completely understood. We have recently demonstrated that the ptxR upstream region contains potential binding sites for multiple regulators, including the virulence factor regulator Vfr. In this study, we identified within the ptxR upstream region, a 25 bp sequence to which Vfr specifically binds. The sequence is located 20–44 (32.5) bp 5′ of the ptxR P2 promoter, and overlaps a potential binding site for the iron-starvation sigma factor PvdS. We also show that, throughout the growth cycle, deletion of vfr reduces ptxR expression from the P2 promoter in the P. aeruginosa strain PAO1 by four- to eightfold, but does not affect ptxR expression from P1. Further, loss of Vfr eliminates the PtxR-induced enhancement in the synthesis of exotoxin A and the metalloproteinase LasB. Our results suggest that Vfr modulates toxA and lasB expression in PAO1 through PtxR. A model defining the relationships between these different genes is presented.

Adherent and invasive mucosa-associated Escherichia coli have been implicated in the pathogenesis of colon cancer and inflammatory bowel diseases. It has been reported that such isolates share features of extraintestinal E. coli (ExPEC) and particularly uropathogenic E. coli (UPEC). We used suppression subtractive hybridization (SSH) to subtract the genome of E. coli K-12 from that of a colon cancer mucosal E. coli isolate. Of the subtracted sequences, 53 % were present in the genomes of one or more of three sequenced UPEC strains but absent from the genome of an enterohaemorrhagic E. coli (EHEC) strain. Of the subtracted sequences, 80 % matched at least one UPEC genome, whereas only 4 % were absent from the UPEC genomes but present in the genome of the EHEC strain. A further genomic subtraction against the UPEC strain 536 enriched for sequences matching mobile genetic elements, other ExPEC strains, and other UPEC strains or commensals, rather than strains associated with gastrointestinal disease. We analysed the distribution of selected subtracted sequences and UPEC-associated pathogenicity islands (PAIs) amongst a panel of mucosa-associated E. coli isolated from colonoscopic biopsies of patients with colon cancer, patients with Crohn's disease and controls. This enabled us to identify a group of isolates from colon cancer (30–40 %) carrying multiple genes previously categorized as UPEC-specific and implicated in virulence.

Bacterial alkaline phosphatases (PhoA) hydrolyse phosphate-containing substrates to provide the preferred phosphorus source inorganic phosphate (Pi). Campylobacter jejuni does not contain a typical PhoA homologue but contains a phosphatase that is regulated by the two-component system PhosS/PhosR. Here we describe the characterization of the enzyme, its secretion pathway and its function in the bacterium's biology. Phosphatase assays showed that the enzyme utilizes exclusively phosphomonoesters as a substrate, requires Ca2+ for its activity, and displays maximum activity at a pH of 10. Gene disruption revealed that it is the sole alkaline phosphatase in C. jejuni. The protein contained a twin-arginine motif (RR) at its N terminus, typical of substrates of the Tat secretion system. Substitution of the twin-arginine residues showed that they are essential for enzyme activity. C. jejuni genome analysis indicated the presence of four ubiquitously expressed Tat components that may form a functional Tat secretion system as well as 11 putative Tat substrates, including the alkaline phosphatase (PhoACj) and the nitrate reductase NapA. Inactivation of tatC caused defects in both PhoACj and NapA activity as well as a reduction in bacterial growth that were all restored by complementation in trans with an intact tatC copy. The atypical overall features of the PhoACj compared to Escherichia coli PhoA support the existence in prokaryotes of a separate group of Tat-dependent alkaline phosphatases, classified as the PhoX family.

Carbon starvation is a significant stress encountered by the opportunistic fungal pathogen Candida albicans, and mutations in several pathways required to assimilate non-fermentable carbon sources attenuate virulence. These pathways – β-oxidation, the glyoxylate cycle and gluconeogenesis – are compartmentalized in the fungal cell between the peroxisome, mitochondria and cytosol; thus, the cell must transport key intermediates between these organelles. Transport of acetyl-CoA, a particularly important intermediate of carbon metabolism, is catalysed by membrane-associated carnitine acetyltransferases (CATs). We report here the characterization of the three predicted CAT genes in C. albicans, CTN1, CTN2 and CTN3. Strains lacking CTN1 or CTN2 were unable to grow on ethanol or acetate as sole carbon source; additionally, citrate was utilized poorly (Δctn2) or not at all (Δctn1) and the Δctn2 mutant failed to grow on fatty acids as well. In contrast, deletion of CTN3 had no observable phenotype. All three genes were upregulated in the presence of non-fermentable carbon sources and after macrophage phagocytosis. CTN1 and CTN3 were able to complement the corresponding Saccharomyces cerevisiae Δyat1 and Δyat2 mutants. However, these mutants had no obvious attenuation in virulence in a mouse model of disseminated candidiasis, in contrast to other carbon metabolism mutants. These findings extend our understanding of nutrient stress in vivo and in vitro and the contribution of metabolic pathways to virulence in C. albicans.

Proteins that are covalently linked to the skeletal polysaccharides of the cell wall of Candida albicans play a major role in the colonization of the vaginal mucosal surface, which may result in vaginitis. Here we report on the variability of the cell-wall proteome of C. albicans as a function of the ambient O2 concentration and iron availability. For these studies, cells were cultured at 37 °C in vagina-simulative medium and aerated with a gas mixture consisting of 6 % (v/v) CO2, 0.01–7 % (v/v) O2 and N2, reflecting the gas composition in the vaginal environment. Under these conditions, the cells grew exclusively in the non-hyphal form, with the relative growth rate being halved at ∼0.02 % (v/v) O2. Using tandem MS and immunoblot analysis, we identified 15 covalently linked glycosylphosphatidylinositol (GPI) proteins in isolated walls (Als1, Als3, Cht2, Crh11, Ecm33, Hwp1, Pga4, Pga10, Phr2, Rbt5, Rhd3, Sod4, Ssr1, Ywp1, Utr2) and 4 covalently linked non-GPI proteins (MP65, Pir1, Sim1/Sun42, Tos1). Five of them (Als3, Hwp1, Sim1, Tos1, Utr2) were absent in cells grown in rich medium. Immunoblot analysis revealed that restricted O2 availability resulted in higher levels of the non-GPI protein Pir1, a putative β-1,3-glucan cross-linking protein, and of the GPI-proteins Hwp1, an adhesion protein, and Pga10 and Rbt5, which are involved in iron acquisition. Addition of the iron chelator ferrozine at saturating levels of O2 resulted in higher cell wall levels of Hwp1 and Rbt5, suggesting that the responses to hypoxic conditions and iron restriction are related.

Tropheryma whipplei, the causative agent of Whipple's disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipple's disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipple's disease associated with additional manifestations, six relapses of classic Whipple's disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipple's disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipple's disease may, however, be useful in epidemiological studies.

Most Aggregatibacter actinomycetemcomitans strains express relatively low levels of leukotoxin, encoded by the orfA–ltxCABD operon. However, several strains isolated from patients with localized aggressive periodontitis are hyperleukotoxic and transcribe the ltx operon at high levels. These strains possess a copy of IS1301 in the ltx promoter and previous studies have suggested that the presence of the insertion sequence increases ltx transcription by uncoupling a cis-acting negative regulator of ltx expression from the basal elements of the ltx promoter. However, we now report that replacing IS1301 with an equal length of random sequence has little effect on transcriptional activity of the ltx promoter, suggesting that the physical displacement of the negative regulatory element does not contribute to the hyperleukotoxic phenotype of IS1301-containing strains. Instead, we show that a −10-like element upstream of the transposase ORF of IS1301 is required for increased transcriptional activity of the ltx promoter. Site-specific mutation of the −10 sequence, or reversing the orientation of IS1301 relative to the basal ltx promoter elements, reduced transcriptional activity to levels exhibited by the native ltx promoter. However, no increase in transcription was observed when IS1301 was recombinantly inserted into a ltx promoter that contained a truncated copy of orfA, suggesting that an intact orfA may also be required for IS1301-mediated induction of ltxCABD. Therefore, to determine if orfA functions as a regulator of ltx expression, three independent ltx-promoter–lacZ-reporter constructs containing frameshift mutations in orfA were analysed. Each exhibited significantly lower expression of β-galactosidase than the control reporter with intact orfA. In addition, OrfA protein was shown, by mobility shift electrophoresis, to interact with the ltx promoter at or downstream of the −35 sequence. These results suggest that a potential transposase promoter and the OrfA polypeptide may modulate leukotoxin expression in hyperleukotoxic A. actinomycetemcomitans strains containing IS1301.