- Volume 151, Issue 4, 2005

Genes encoding eukaryotic-type protein kinases and phosphatases are present in many bacterial genomes. An ORF encoding a polypeptide with homology to protein phosphatases 2C (PP2Cs) was identified in the genomes of Salmonella enterica serovar Typhi strains CT18 and Ty2. This protein, termed PrpZ, is the first PP2C to be identified in enterobacteria. Analysis of the amino acid sequence revealed two distinct domains: the N-terminal segment containing motifs of the catalytic domain of PP2Cs and the C-terminal segment with unknown function. PrpZ was expressed in Escherichia coli as a histidine-tagged fusion protein (PrpZHis) and the purified protein was analysed for its ability to dephosphorylate various substrates. Using p-nitrophenyl phosphate as a substrate, optimal PrpZHis activity was observed at pH 9·5, with a strong preference for Mn2+ over Mg2+. Activity of PrpZHis was inhibited by EDTA, sodium fluoride, sodium phosphate and sodium pyrophosphate but unaffected by okadaic acid, indicating that PrpZ is a PP2C. Using synthetic phosphopeptides as substrates, PrpZHis could hydrolyse phosphorylated serine, threonine or tyrosine residues, with the highest catalytic efficiency (k cat/K m) for the threonine phosphopeptide. With phosphorylated myelin basic protein (MBP) as the substrate, Mn2+ was only twofold more efficient than Mg2+ in stimulating PrpZHis activity at pH 8·0. The ability of PrpZHis to remove the phosphoryl group from phosphotyrosine residues was confirmed by measuring the release of inorganic phosphate from phospho-Tyr MBP. Together, these data indicate that PrpZ has all the features of a PP2C with dual substrate specificity in vitro.

Salmonella enterica degrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B12 (adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization (pdu) are found in a large contiguous cluster, the pdu locus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B12 (cyanocobalamin; CNCbl) to AdoCbl and that the pduO gene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here, in vitro evidence is presented that the pduS gene encodes a bifunctional cobalamin reductase that catalyses two reductive steps needed for the conversion of CNCbl into AdoCbl. The PduS enzyme was produced in high levels in Escherichia coli. Enzyme assays showed that cell extracts from the PduS expression strain reduced cob(III)alamin (hydroxycobalamin) to cob(II)alamin at a rate of 91 nmol min−1 mg−1 and cob(II)alamin to cob(I)alamin at a rate of 7·8 nmol min−1 mg−1. In contrast, control extracts had only 9·9 nmol min−1 mg−1 cob(III)alamin reductase activity and no detectable cob(II)alamin reductase activity. Thus, these results indicated that the PduS enzyme is a bifunctional cobalamin reductase. Enzyme assays also showed that the PduS enzyme reduced cob(II)alamin to cob(I)alamin for conversion into AdoCbl by purified PduO adenosyltransferase. Moreover, studies in which iodoacetate was used as a chemical trap for cob(I)alamin indicated that the PduS and PduO enzymes physically interact and that cob(I)alamin is sequestered during the conversion of cob(II)alamin to AdoCbl by these two enzymes. This is likely to be important physiologically, since cob(I)alamin is extremely reactive and would need to be protected from unproductive by-reactions. Lastly, bioinformatic analyses showed that the PduS enzyme is unrelated in amino acid sequence to enzymes of known function currently present in GenBank. Hence, results indicate that the PduS enzyme represents a new class of cobalamin reductase.

Heat-labile enterotoxin, a major virulence determinant of enterotoxigenic Escherichia coli, is encoded by the eltAB operon. To elucidate the molecular mechanism by which the heat-stable nucleoid-structural (H-NS) protein controls transcription of eltAB, the authors constructed an eltAB–lacZ transcriptional fusion and performed β-galactosidase analysis. The results showed that H-NS protein exerts fivefold repression on transcription from the eltAB promoter at 37 °C and 10-fold repression at 22 °C. Two silencer regions that were required for H-NS-mediated repression of eltAB expression were identified, both of which were located downstream of the start site of transcription. One silencer was located between +31 and +110, the other between +460 and +556, relative to the start site of transcription, and they worked cooperatively in repression. DNA sequences containing the silencers were predicted to be curved by in silico analysis and bound H-NS protein directly in vitro. Repression of eltAB transcription by H-NS was independent of promoter strength, and the presence of H-NS protein did not affect promoter opening in vitro, indicating that repression was achieved by inhibiting promoter clearance or blocking transcription elongation, probably via DNA looping between the two silencers.

Manganese serves an important function in Lactobacillus plantarum in protection against oxidative stress and this bacterium can accumulate Mn2+ up to millimolar levels intracellularly. Although the physiological role of Mn2+ and the uptake of this metal ion have been well documented, the only uptake system described so far for this bacterium is the Mn2+- and Cd2+-specific P-type ATPase (MntA). Recently, the genome of L. plantarum WCFS1 has been sequenced allowing in silico detection of genes potentially encoding Mn2+ transport systems, using established microbial Mn2+ transporters as the query sequence. This genome analysis revealed that L. plantarum WCFS1 encodes, besides the previously described mntA gene, an ABC transport system (mtsCBA) and three genes encoding Nramp transporters (mntH1, mntH2 and mntH3). The expression of three (mtsCBA, mntH1 and mntH2) of the five transport systems was specifically derepressed or induced upon Mn2+ limitation, supporting their role in Mn2+ homeostasis in L. plantarum. However, in contrast to previous reports, mntA expression remains below detection levels in both Northern and real-time RT-PCR analysis in both Mn2+ excess and starvation conditions. Growth of WCFS1 derivatives mutated in mntA, mtsA or mntH2, or both mtsA and mntH2 appears unaffected under Mn2+ excess or Mn2+ limitation. Moreover, intracellular Mn2+ concentrations remained unaltered in these mutants compared to the wild-type. This may suggest that this species is highly adaptive in response to inactivation of these genes or, alternatively, that other transporters that have not yet been identified as Mn2+ transporters in bacteria are involved in Mn2+ homeostasis in L. plantarum.

Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpC/gp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure.

The serotype F phage π42 of Staphylococcus aureus is a triple-converting bacteriophage that encodes the staphylokinase gene (sak) and the enterotoxin A gene (entA). Lysogeny results in loss of expression of the chromosomal β-haemolysin gene (hlb) (negative conversion), the expression of staphylokinase and enterotoxin A (positive conversion), and the acquisition of resistance to lysis by all 23 phages of the International Basic Set (IBS) of S. aureus typing phages. Until this study, the basis of π42 resistance to lysis by exogenous phages was unknown. The authors report here that phage π42 encodes a restriction–modification (R–M) system, termed Sau42I, adjacent to and in the same orientation to the phage integrase gene int. The genes encoding Sau42I were cloned and sequenced, and found to consist of two overlapping reading frames, ORF S (specificity) and ORF RM (restriction–modification), in the same orientation. The ORFs share a high degree of DNA and amino acid sequence homology with the previously characterized BcgI R–M system of Bacillus coagulans. Expression of the cloned Sau42I ORF S and ORF RM in S. aureus 80CR3 transformants from a plasmid vector conferred resistance to lysis by all 23 IBS phages. Similarly, transformants of S. aureus RN4220 harbouring recombinant plasmids containing both ORFs were resistant to lysis by the IBS typing phages. However, transformants harbouring plasmids encoding either ORF S or ORF RM were susceptible to lysis by the IBS phages, and they had the same phage-susceptibility pattern as the respective parental isolates. In vitro analysis of crude and partially purified extracts of S. aureus transformants harbouring both the π42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg2+ and S-adenosylmethionine in order to function, and activity is optimized at pH 8, although the precise recognition sequence has yet to be determined. The findings of this study confirm that π42 is a quadruple-converting phage, believed to be the first described for S. aureus, and show that it encodes a novel R–M system termed Sau42I.

Two neurotoxic alkaloids, anatoxin-a and its homologue homoanatoxin-a, were purified from the filamentous cyanobacteria Oscillatoria sp. strain 193 (PCC 9240) and Oscillatoria formosa NIVA CYA-92 (PCC 10111), respectively, and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors, using post-synaptic membrane fractions of Torpedo electric tissue. Inhibition was concentration dependent, with a K i of 5·4±1·1×10−8 M for anatoxin-a and 7·4±0·9×10−8 M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains, representative of 13 genera, were analysed. Five strains of the genus Oscillatoria, hitherto unknown for their toxicity, inhibited bungarotoxin binding. GC/MS revealed that Oscillatoria sp. strains PCC 6407, PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a, whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. Oscillatoria sp. strain PCC 6506, an isolate co-identic with strain PCC 9029, also produced both neurotoxins, but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence.

The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal medium supplemented with elevated copper concentrations (as copper-glycine) has been analysed using whole-genome oligonucleotide microarrays. At 750 μM copper-glycine, the expression of both the cue and cus copper-export systems is evident. At near-lethal copper concentrations (2 mM copper-glycine), the expression of these two regulons increases significantly. Other regulons with increased transcription in response to elevated concentrations of copper-glycine include those for the superoxide stress response, iron homeostasis, and envelope stress. Furthermore, a variety of ORFs with decreased expression in response to increased copper-glycine has been identified, including the zinc ABC transporter and genes involved in the chemotactic response.

Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1×101 to 2×104 c.f.u. (μg introduced transposon DNA)−1. The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.

The amplification of an internal fragment of the hdcA gene for histidine decarboxylase in Lactobacillus buchneri showed the gene to be located on the bacterial chromosome. Reverse PCR was then used to amplify both it and its adjacent genes. The histidine decarboxylase cluster was found to be composed of four genes: hdcC (expressed in Lactococcus lactis, the product of which is located in the membrane, suggesting it to be a histidine/histamine antiporter), hdcA (which encodes histidine decarboxylase), hdcB (of unknown function but co-transcribed as bicistronic mRNA together with hdcA) and hisS (the only copy of a gene encoding a histidyl-tRNA synthetase in Lb. buchneri). The expression of hisS depends on the histidine concentration of the growth medium, and it can be transcribed as monocistronic or hdcA–hdcB–hisS polycistronic mRNA.

The gene encoding yeast-enhanced green fluorescent protein (GFP) was placed under control of ALS gene promoters in Candida albicans. The PALS-GFP reporter strains were validated using various techniques including a new real-time RT-PCR assay to quantify ALS gene expression. The PALS-GFP reporter strains were grown in media that promoted yeast or germ tube forms, and the resulting fluorescence was measured by flow cytometry. In addition to results that indicate differences in ALS gene expression due to growth medium, growth stage and developmental programme, new data show large differences in transcriptional level among the ALS genes. Expression of ALS1 was associated with transfer of the PALS1-GFP strain to fresh growth medium. ALS3 expression increased markedly when germ tubes were visible microscopically and ALS7 expression exhibited a transient peak between 2 and 3 h following inoculation into fresh YPD medium. Transcription from the ALS1 and ALS3 promoters was strongest among those tested and contrasted markedly with the weaker promoter strength at the ALS5, ALS6, ALS7 and ALS9 loci. These weaker transcriptional responses were also observed using real-time RT-PCR measurements on wild-type C. albicans cells. Assuming a positive correlation between transcriptional level and protein production, these results suggest that some Als proteins are abundant on the C. albicans cell surface while others are produced at a much lower level.

HWP1 encodes an adhesin of Candida albicans and has been implicated in filamentation and virulence. URA3, an often-used transformation selection marker, is apparently incorrectly expressed when integrated at the HWP1 locus, which results in an attenuated virulence phenotype. Expression of URA3 is compromised by ectopic integration at other loci as well. In contrast, prior studies from the authors' laboratory had demonstrated that the filamentation deficiency and attenuated virulence of hwp1Δ mutants were fully restored in rescued strains in which URA3 was integrated at the HWP1 locus. This discrepancy prompted a reinvestigation of these mutants. A series of congenic strains were constructed which demonstrated that the filamentation and virulence defects of a homozygous hwp1Δ mutant could be rescued without introduction of a functional HWP1 allele. Despite the absence of detectable differences in URA3 expression, analysis of suppressor mutations suggested that reduced URA3 expression gave rise to the mutant phenotypes. Several independent spontaneous suppressor mutations that restored filamentation to strains of genotype hwp1Δ : : hisG-URA3-hisG/hwp1Δ : : hisG had acquired a tandem duplication of the hisG-URA3-hisG marker cassette. The hwp1 null mutant and rescued strains differed by the presence or absence of flanking hisG sequence. Substitution of the hisG-URA3-hisG insert of the hwp1 null mutant with URA3 alone largely rescued the filamentation and virulence phenotypes. The presence of a single copy of hisG adjacent to URA3 had no effect. It is concluded that flanking direct repeats of hisG, present as part of a recyclable disruption cassette, negatively influenced URA3 expression and are responsible for the previously reported phenotypes of the hwp1 mutants.