- Volume 154, Issue 1, 2008

Urinary tract infection (UTI) is the most common infection in patients with indwelling urinary catheters, and bacterial biofilm formation is a major problem in this type of infection. Escherichia coli is responsible for the large majority of UTIs. Free iron is strictly limited in the human urinary tract and there is fierce competition between the host and infectious bacteria for this essential metal. Urinary tract infectious E. coli have highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding the yersiniabactin receptor, is one of the most upregulated genes in biofilm; it was upregulated 63-fold in the E. coli UTI strain VR50. FyuA was found to be highly important for biofilm formation in iron-poor environments such as human urine. Mutants in fyuA show aberrant biofilm formation and the cells become filamentous; a VR50fyuA mutant showed a 92 % reduction in biofilm formation in urine flow-cell chambers compared with the wild-type. The FyuA/yersiniabactin system is known to be important for virulence. Here we demonstrate a direct link between FyuA and biofilm formation in iron-poor environments. We also show that the availability of iron greatly influences UTI strains' ability to form biofilm.

Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx2 variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx2-vhb variant, which was frequently associated with stx2 , stx2-vha or stx2c . Levels of stx2 and stx2-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx2 variant than in the remaining strains of seropathotype C. The stx2-vhb genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx2 -related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx2 -related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx2 gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

The conjugative plasmid pOLA52, which confers resistance to olaquindox and other antimicrobial agents through a multidrug efflux pump, was investigated for its ability to promote biofilm formation in Escherichia coli. Screening of a transposon-mutagenized pOLA52 clone library revealed several biofilm-deficient mutants, which all mapped within a putative operon with high homology to the mrkABCDF operon of Klebsiella pneumoniae, where these genes are responsible for type 3 fimbriae expression, attachment to surfaces and biofilm formation. Biofilm formation in microtitre plates and in urinary catheters of clones containing pOLA52 with a disrupted putative mrk operon was reduced by more than 100-fold and 2-fold, respectively, compared to mutants with an intact mrk operon. The conjugative transfer rate of pOLA52 was also significantly lower when the mrk operon was disrupted. Through reverse transcriptase analysis, it was demonstrated that the genes contained in the putative mrk operon were linked and likely to be expressed as a single operon. Immunoblotting with type 3 fimbriae (MrkA)-specific antibodies further verified expression of type 3 fimbriae. When transferred to other, potentially pathogenic, members of the family Enterobacteriaceae, including Klebsiella pneumoniae, Salmonella Typhimurium, Kluyvera sp. and Enterobacter aerogenes, pOLA52 facilitated increased biofilm formation. pOLA52 is believed to represent the first example of a conjugative plasmid encoding type 3 fimbriae, resulting in enhanced conjugation frequencies and biofilm formation of the plasmid-harbouring strain.

DsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PilQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.

‘Pseudomonas butanovora’ uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C2–C9 n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative σ 54-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C2–C5 n-alkanes and showed little to no growth on C6–C8 n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the ‘P. butanovora’ bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C2–C8 carbon sources. In acetate- or lactate-grown cells, C2–C8 n-alcohols failed to induce β-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced ∼45 % of the β-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C2–C5 n-alcohols induced β-galactosidase activity, whereas C7 and C8 n-alcohols did not. BmoR may act as a σ 54-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. ≤C6). The bmoG-deficient mutant did not grow on C2–C8 n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.

Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 °C compared to 37 °C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 °C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 °C. Another category of genes more highly expressed at 23 °C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 °C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 °C. Increased expression at 23 °C compared to 37 °C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 °C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 °C and viability at 4 °C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 °C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 °C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 °C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response – the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

Formaldehyde is an intermediate formed during the metabolism of methanol or other methylated compounds. Many Gram-negative bacteria generate formaldehyde from methanol via a periplasmic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in which the α subunit of an α 2 β 2 tetramer has catalytic activity. The genome of the facultative formaldehyde-oxidizing bacterium Rhodobacter sphaeroides encodes XoxF, a homologue of the catalytic subunit of a proposed PQQ-containing dehydrogenase of Paracoccus denitrificans. R. sphaeroides xoxF is part of a gene cluster that encodes periplasmic c-type cytochromes, including CycI, isocytochrome c 2 and CycB (a cyt c 553i homologue), as well as adhI, a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), and gfa, a homologue of a glutathione–formaldehyde activating enzyme (Gfa). To test the roles of XoxF, CycB and Gfa in formaldehyde metabolism by R. sphaeroides, we monitored photosynthetic growth with methanol as a source of formaldehyde and whole-cell methanol-dependent oxygen uptake. Our data show that R. sphaeroides cells lacking XoxF or CycB do not exhibit methanol-dependent oxygen uptake and lack the capacity to utilize methanol as a sole photosynthetic carbon source. These results suggest that both proteins are required for formaldehyde metabolism. R. sphaeroides Gfa is not essential to activate formaldehyde, as cells lacking gfa are capable of both methanol-dependent oxygen uptake and growth with methanol as a photosynthetic carbon source.

Heat stress caused rapid and severe inhibition of photosynthesis and nitrate reduction in nitrate-supplemented cultures of the cyanobacterium Anabaena sp. strain L-31, compared to nitrogen-fixing cultures. Anabaena strains harbour two hsp60 family genes, groEL and cpn60, respectively encoding the 59 kDa GroEL and 61 kDa Cpn60 chaperonin proteins. Of these two Hsp60 chaperonins, GroEL was strongly induced during heat stress, irrespective of the nitrogen status of the cultures, but Cpn60 was rapidly repressed and degraded in heat-stressed nitrate or ammonium-supplemented cultures. The recovery of photosynthesis, nitrate assimilation and growth in heat-stressed, nitrate-supplemented cultures were preceded by resynthesis and restoration of cellular Cpn60 levels. Glutamine synthetase activity, although adversely affected by prolonged heat stress, was not dependent on either the nitrogen status or Cpn60 levels during heat stress. Overexpression of the Cpn60 protein in the closely related Anabaena sp. strain PCC7120 conferred significant protection from heat stress to growth, photosynthesis and nitrate reduction in the recombinant strain. The data favour a role for Cpn60 in carbon and nitrogen assimilation in Anabaena.

Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribosome biogenesis. During growth in shake flasks, a mutant deleted for SFP1 shows a small size phenotype and a reduced growth rate. We characterized the behaviour of an sfp1Δ mutant compared to an isogenic reference strain growing in chemostat cultures at the same specific growth rate. By studying glucose (anaerobic)- and ethanol (aerobic)-limited cultures we focused specifically on nutrient-dependent effects. Major differences in the genome-wide transcriptional profiles were observed during glucose-limited growth. In particular, Sfp1 appeared to be involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Flow cytometric analyses revealed size defects for the mutant under both growth conditions. Our results suggest that Sfp1 plays a role in transcriptional and cell size control, operating at two different levels of the regulatory network linking growth, metabolism and cell size.