- Volume 149, Issue 11, 2003

The full sequence of plasmid p250, isolated from Haemophilus paragallinarum strain HP250, has been obtained. The plasmid contains seven ORFs: a putative integrase, a putative replication protein (repB) and five ORFs similar to those from the haemocin (bacteriocin) hmcDCBAI operon from Haemophilus influenzae. Of 19 other non-plasmid-containing H. paragallinarum strains screened (11 serovar reference strains and 8 field isolates), 17 strains produced haemocin and were resistant to killing by strain HP250. These strains, unlike strain HP250, have a chromosomally encoded haemocin operon. A number of other members of the family Pasteurellaceae were tested for haemocin sensitivity. Pasteurella avium, Pasteurella volantium and Pasteurella species A, all non-pathogenic bacteria found in the respiratory tract of chickens suffering from respiratory diseases, were sensitive to H. paragallinarum haemocin. However, amongst the pathogenic Pasteurellaceae, 50 % of P. multocida isolates and all five isolates of Pasteurella haemolytica tested were sensitive to the haemocin. Given the prevalence of haemocin production in H. paragallinarum strains, it may play a role in aiding colonization by inhibiting other Gram-negative bacteria that are associated with the respiratory tract in chickens. The origin of replication from plasmid p250 has been used to generate an Escherichia coli–H. paragallinarum shuttle vector which may be useful in genetically manipulating H. paragallinarum.

An aquaporin gene (aqpX) was previously detected in the pathogenic bacterium Brucella abortus. Earlier studies showed that AqpX mediated rapid and large water fluxes in both directions in response to sudden osmotic up- or downshifts. Here, to study the role and the expression of the aqpX gene in B. abortus, an aqpX null mutant was constructed using an aqpX : : lacZ gene fusion. This mutant showed no significant difference in growth rate compared to the wild-type strain when grown in rich and minimal media, demonstrating that disruption of the aqpX gene was not lethal for B. abortus. The role of the B. abortus AqpX water channel was investigated by exposing the cells to hypo- and hyperosmolar conditions. While in hyperosmolar environments the growth rate of the knockout mutant was not affected, in hypo-osmolar conditions this mutant showed reduced viability after 50 h of growth. β-Galactosidase assays and RT-PCR revealed that aqpX gene expression and the amount of aqpX mRNA were markedly increased in hyperosmolar conditions. Moreover, B. abortus aqpX expression levels were enhanced during the mid-exponential phase of growth. These results indicated that the expression of aqpX was regulated during the growth curve and induced in hyperosmolar conditions. This report is believed to be the first example of the induction of a bacterial aquaporin in hypertonic conditions.

The Mycobacterium tuberculosis complex includes Mycobacterium bovis, which causes tuberculosis in most mammals, including humans. In previous work, it was shown that M. bovis ATCC 35721 has a mutation in its principal sigma factor gene, sigA, causing a single amino acid change affecting binding of SigA with the accessory transcription factor WhiB3. ATCC 35721 is avirulent when inoculated subcutaneously into guinea pigs but can be restored to virulence by integration of wild-type sigA to produce M. bovis WAg320. Subsequently, it was surprising to discover that WAg320 was not virulent when inoculated intratracheally into the Australian brushtail possum (Trichosurus vulpecula), a marsupial that is normally very susceptible to infection with M. bovis. In this study, an in vivo complementation approach was used with ATCC 35721 to produce M. bovis WAg322, which was virulent in possums, and to identify the virulence-restoring gene, phoT. There are two point deletions in the phoT gene of ATCC 35721 causing frameshift inactivation, one of which is also in the phoT of BCG. Knockout of phoT from ATCC 35723, a virulent strain of M. bovis, produced M. bovis WAg758, which was avirulent in both guinea pigs and possums, confirming that phoT is a virulence gene. The effect on virulence of mode of infection versus animal species susceptibility was investigated by inoculating all the above strains by aerosol into guinea pigs and mice and comparing these to the earlier results. Characterization of PhoT indicated that it plays a role in phosphate uptake at low phosphate concentrations. At least in vitro, this role requires the presence of a wild-type sigA gene and appears separate from the ability of phoT to restore virulence to ATCC 35721. This study shows the advantages of using different animal models as tools for the molecular biological investigation of tuberculosis virulence.

Using molecular methods the authors have studied mycobacterial DNA taken from a 19th century victim of tuberculosis. This was the case from which Robert Koch first isolated and cultured the organism responsible for tuberculosis. The mycobacteria were preserved within five glass culture tubes as abundant bacterial colonies on slopes of a gelatinous culture medium of unknown composition. Originally presented by Koch to surgical laryngologist Walter Jobson Horne in London in 1901, the relic has, since 1983, been in the care of the Royal College of Surgeons of England. Light and electron microscopy established the presence of acid-fast mycobacteria but showed that morphological preservation was generally poor. Eleven different genomic loci were successfully amplified by PCR. This series of experiments confirmed that the organisms were indeed Mycobacterium tuberculosis and further showed that the original strain was in evolutionary terms similar to ‘modern’ isolates, having undergone the TB D1 deletion. Attempts to determine the genotypic group of the isolate were only partially successful, due in part to the degraded nature of the DNA and possibly also to a truncation in the katG gene, which formed part of the classification scheme. Spoligotyping resulted in amplification of DR spacers consistent with M. tuberculosis but with discrepancies between independent extracts, stressing the limitations of this typing method when applied to poorly preserved material.

To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9·5 % an intermediate infection, and 9·5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.

Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.

Poly(ethylene oxide) (PEO)-brushes are generally recognized as protein-repellent surfaces, and although a role in discouraging microbial adhesion has been established for some strains and species, no study exists on the effects of PEO-brushes on a large variety of bacterial and yeast strains. In this paper, a PEO-brush has been covalently attached to glass and silica by reaction in a polymer melt. Subsequently, the presence of a PEO-brush was demonstrated using contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. For five bacterial (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa) and two yeast strains (Candida albicans and Candida tropicalis), adhesion to PEO-brushes was compared with adhesion to bare glass in a parallel plate flow chamber. The initial deposition rates of Sta. epidermidis, Sta. aureus and Str. salivarius to glass were relatively high, between 2400 and 2600 cm−2 s−1, while E. coli and P. aeruginosa deposited much more slowly. The initial deposition rates of the yeasts to glass were 144 and 444 cm−2 s−1 for C. albicans GB 1/2 and C. tropicalis GB 9/9, respectively. Coating of the glass surface with a PEO-brush yielded more than 98 % reduction in bacterial adhesion, although for the more hydrophobic P. aeruginosa a smaller reduction was observed. For both yeast species adhesion suppression was less effective than for the bacteria and here too the more hydrophobic C. tropicalis showed less reduction than the more hydrophilic C. albicans. The PEO-brush had a thickness of 22 nm in water, as inferred from ellipsometry. Assuming that on bare glass the adhered micro-organisms are positioned only a few nanometers away from the surface and that the brush keeps them at a distance of 22 nm, it is calculated that the brush yields a sevenfold attenuation of the Lifshitz–Van der Waals attraction to the surface between the micro-organisms and the surface. Decreased Lifshitz–van der Waals attraction may be responsible for the suppression of the microbial adhesion observed.

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, a starvation of combined nitrogen induces differentiation of heterocysts, cells specialized in nitrogen fixation. How do filaments perceive the limitation of the source of combined nitrogen, and what determines the proportion of heterocysts? In cyanobacteria, 2-oxoglutarate provides a carbon skeleton for the incorporation of inorganic nitrogen. Recently, it has been proposed that the concentration of 2-oxoglutarate reflects the nitrogen status in cyanobacteria. To investigate the effect of 2-oxoglutarate on heterocyst development, a heterologous gene encoding a 2-oxoglutarate permease under the control of a regulated promoter was expressed in Anabaena sp. PCC 7120. The increase of 2-oxoglutarate within cells can trigger heterocyst differentiation in a subpopulation of filaments even in the presence of nitrate. In the absence of a source of combined nitrogen, it can increase heterocyst frequency, advance the timing of commitment to heterocyst development and further increase the proportion of heterocysts in a patS mutant. Here, it is proposed that the intracellular concentration of 2-oxoglutarate is involved in the determination of the proportion of the two cell types according to the carbon/nitrogen status of the filament.