- Volume 150, Issue 9, 2004

Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers ( Fabret et al., 1999 , J Bacteriol 181, 1975–1983) to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.

Living organisms display large differences in stress resistance throughout their life cycles. To study the coordinated regulation of development and stress responses in exponentially growing yeast, mutants that displayed elevated heat-shock resistance at this stage were screened for. Here, two new mutant alleles of CDC25 in Saccharomyces cerevisiae, cdc25-21 and cdc25-22, are described. During exponential growth in glucose at 25 °C, these mutants are resistant to heat, oxidative, osmotic and ionic shock, accumulate stress-protein transcripts, show slow growth rates, thick cell walls and glycogen hyperaccumulation and lack cAMP signalling in response to glucose. Genetic and cellular analyses revealed that the stationary-phase phenotypes of cdc25-21 and cdc25-22 mutants are not due to entrance to a G0 state during exponential growth, but are the result of a prolonged G1 phase. It was found that, in the W303 background, CDC25 is dispensable for growth in glucose media. However, CDC25 is essential for growth in galactose, in non-fermentable carbon sources and under continuous incubation at 38 °C. In conclusion, the function of the catalytic, C-terminal domain of Cdc25p is not only important for fermentative growth, but also for growth in non-fermentable carbon sources and to trigger galactose derepression.

The subcellular localization of membrane proteins in Bacillus subtilis was examined by using fluorescent protein fusions. ATP synthase and succinate dehydrogenase were found to localize within discrete domains on the membrane rather than being homogeneously distributed around the cell periphery as expected. Dual labelling of cells indicated partial colocalization of ATP synthase and succinate dehydrogenase. Further analysis using an ectopically expressed phage protein gave the same localization patterns as ATP synthase and succinate dehydrogenase, implying that membrane proteins are restricted to domains within the membrane. 3D reconstruction of images of the localization of ATP synthase showed that domains were not regular and there was no bias for localization to cell poles or any other positions. Further analysis revealed that this localization was highly dynamic, but random, implying that integral membrane proteins are free to diffuse two-dimensionally around the cytoplasmic membrane.

The RNA polymerase core associated with σ S transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by σ D-associated RNA polymerase. A mutation in the gene that encodes σ S, rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of σ S inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by σ S. In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by σ S. The effect of σ S on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position −13 is important for the positive effect of σ S on pst. The interpretation of these observations is based on the competition between σ S and σ D for the binding to the core RNA polymerase.

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes – nmb0278, nmb0294 and nmb0407 – encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress. Evidence was found that this process is regulated at the transcriptional level. S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0·75 M NaCl) or high (2·5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA. Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor σ 70, and one (PectA3) might be dependent on the general stress sigma factor σ S. The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters. In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor σ 32 was found upstream of ectB. Transcription from PectA in C. salexigens followed a pattern typical of σ S-dependent promoters, and was reduced by 50 % in an E. coli rpoS background. These data strongly suggest the involvement of the general stress sigma factor σ S in ectABC transcription in C. salexigens. Expression of PectA–lacZ and PectB–lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system. Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid. Moreover, PectA–lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl3). Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.

Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l−1). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l−1. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.

Hypermutable bacterial pathogens exist at surprisingly high prevalence and benefit bacterial populations by promoting adaptation to selective environments, including resistance to antibiotics. Five hundred Haemophilus influenzae isolates were screened for an increased frequency of mutation to resistance to rifampicin, nalidixic acid and spectinomycin: of the 14 hypermutable isolates identified, 12 were isolated from cystic fibrosis (CF) sputum. Analysis by enterobacterial repetitive intergenic consensus (ERIC)-PCR and ribotyping identified eight distinct genetic fingerprints. The hypermutable phenotype of seven of the eight unique isolates was associated with polymorphisms in conserved sites of mutS. Four of the mutant mutS alleles were cloned and failed to complement the mutator phenotype of a mutS : : TSTE mutant of H. influenzae strain Rd KW20. Antibiotic susceptibility testing of the hypermutators identified one β-lactamase-negative ampicillin-resistant (BLNAR) isolate with two isolates producing β-lactamase. Six isolates from the same patient with CF, with the same genetic fingerprint, were clonal by multilocus sequence typing (MLST). In this clone, there was an evolution to higher MIC values for the antibiotics administered to the patient during the period in which the strains were isolated. Hypermutable H. influenzae with mutations in mutS are prevalent, particularly in the CF lung environment, and may be selected for and maintained by antibiotic pressure.

Balamuthia mandrillaris amoebas are recognized as a causative agent of granulomatous amoebic encephalitis, a disease that is usually fatal. They were first recognized when isolated from the brain of a mandrill baboon that died in the San Diego Zoo Wild Life Animal Park. Subsequently, the amoebas have been found in a variety of animals, including humans (young and old, immunocompromised and immunocompetent persons), in countries around the world. Until recently, the amoebas had not been recovered from the environment and their free-living status was in question. The recovery of a Balamuthia amoeba from a soil sample taken from a plant at the home of a child from California, USA, who died of Balamuthia amoebic encephalitis, was reported previously. In a continued investigation, a second amoeba was isolated from soil that was obtained from an outdoor potted plant in a spatially unrelated location. A comparison of these two environmental amoebas that were isolated from different soils with the amoeba that was obtained from the child's clinical specimen is reported here. Included are the isolation procedure for the amoebas, their growth requirements, their immunological response to anti-Balamuthia serum, their sensitivity to a selection of antimicrobials and sequence analysis of their 16S rRNA gene. The evidence is consistent that the amoebas isolated from both soil samples and the clinical isolate obtained from the Californian child are B. mandrillaris.

This research developed a community genome array (CGA) to assess the effects of Acacia angustissima on rumen microbiology. A. angustissima produces non-protein amino acids as well as tannins, which may be toxic to animals, and CGA was used to assess the effects of this plant on the ecology of the rumen. CGAs were developed using a 7·5 cm×2·5 cm nylon membrane format that included up to 96 bacterial genomes. It was possible to separately hybridize large numbers of membranes at once using this mini-membrane format. Pair-wise cross-hybridization experiments were conducted to determine the degree of cross-hybridization between strains; cross-hybridization occurred between strains of the same species, but little cross-reactivity was observed among different species. CGAs were successfully used to survey the microbial communities of animals consuming an A. angustissima containing diet but quantification was not precise. To properly quantify and validate the CGA, Fibrobacter and Ruminococcus populations were independently assessed using 16S rDNA probes to extracted rRNA. The CGA detected an increase in these populations as acacia increased in the diet, which was confirmed by rRNA analysis. There was a great deal of variation among strains of the same species in how they responded to A. angustissima. However, in general Selenomonas strains tended to be resistant to the tannins in the acacia while Butyrivibrio fibrisolvens was sensitive. On the other hand some species, like streptococci, varied. Streptococcus bovis-like strains were sensitive to an increase in acacia in the diet while Streptococcus gallolyticus-like strains were resistant. Strep. gallolyticus has independently been shown to be resistant to tannins. It is concluded that there is significant variation in tannin resistance between strains of the same species. This implies that there are specific molecular mechanisms at play that are independent of the phylogenetic position of the organism.

Proteus mirabilis, a Gram-negative urinary tract pathogen, has two highly homologous, tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a σ 28 promoter, while flaB is a silent allele. Previous studies have demonstrated the presence of a family of hybrid flagellin genes, referred to as flaAB. These genes are composed of the 5′ end of flaA and the 3′ end of flaB, and are produced through excision of the intervening DNA between the two genes. Although the existence of flaAB DNA has been documented, it was not known if transcription of flaAB occurs in wild-type P. mirabilis. In this study, proof of flaAB transcription was obtained from a combination of RNA dot-blots and RT-PCR assays using specific primers and probes for flaAB and flaA. The RNA data were further supported by the demonstration of phenotypic switching of the locus using a FlaAB-detector strain. The results show that flaAB mRNA is transcribed and is 1/64 as abundant as flaA in the population of wild-type cells, suggesting that flaAB constitutes 1·0–1·5 % of the total flagellin message. Nucleotide sequence analysis of flaAB products produced by RT-PCR from the wild-type confirms previous reports of a variable fusion site between flaA and flaB resulting in a hybrid flagellin transcript. These data support the hypothesis that the production of FlaAB is integral to the physiology of P. mirabilis.

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).

The immune response and the anticandidal activity of keratinocytes and polymorphonuclear leukocytes (PMNs) play a key role in host defence against localized Candida albicans infection. An established model of oral candidosis based on reconstituted human oral epithelium (RHE) was supplemented with PMNs to study the effect of these immune cells during experimental oral candidosis. Infection of RHE with C. albicans induced a strong expression of the chemokine interleukin-8 (IL-8) and the cytokine granulocyte-macrophages colony-stimulating factor (GM-CSF), and a moderate stimulation of interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon γ (IFN-γ) and tumour necrosis factor α (TNF-α) by keratinocytes. This immune response was associated with chemoattraction of PMNs to the site of infection, whereas uninfected RHE failed to induce cytokine expression or to attract PMNs. Growth of the pathogen and tissue damage of C. albicans-infected RHE were significantly reduced when PMNs were applied to the apical epithelial surface or when PMNs migrated through a perforated basal polycarbonate filter of the model. Notably, protection against epithelial tissue damage was also observed when PMNs were placed on the basal side of non-perforated filters, which prevented PMN migration into the RHE. Addition of PMNs enhanced a Th1-type immune response (IFN-γ, TNF-α), down-regulated the expression of the Th2-type cytokine interleukin-10 (IL-10), and was associated with protection against Candida-induced tissue damage. This PMN-supplemented model of oral candidosis mimics the in vivo situation, and provides a promising tool for studying the immunological interactions between keratinocytes and C. albicans, as well as the influence of PMNs on C. albicans pathogenesis.

The haploid amoeba Dictyostelium discoideum is a versatile host system for studying cellular aspects of Legionella pathogenicity. Previous studies have shown that the internalization of L. pneumophila leads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth of L. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of L. pneumophila-infected host cells. In contrast to the control experiment with Escherichia coli-containing phagosomes, both proteins decorated the replicative vacuole of L. pneumophila during the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication of L. pneumophila in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of L. pneumophila.

The primary site of infection for Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis is the ciliated respiratory epithelium. Previous studies have implicated adherence of bacteria to cilia, induction of mucus production, induction of ciliostasis and damage to the ciliated epithelium in Bordetella pathogenesis. This paper describes the use of an air-interface organ culture system using canine tracheal tissue infected with B. bronchiseptica to assess the temporal relationship between these pathologies. Ciliostasis occurs very early during the host tissue–pathogen interaction, before mucus production and obvious signs of epithelial damage occur. A B. bronchiseptica bvg mutant does not colonize the organ culture model, induce ciliostasis or cause damage to the epithelial cell layer, but it does induce similar amounts of mucus release as does infection by wild-type bacteria. The authors propose that ciliostasis is a key early event during the B. bronchiseptica–host tissue interaction that abrogates the muco-ciliary defences of the host tissue, renders it susceptible to colonization by the bacteria and allows subsequent damage to the epithelium. The organ culture model described offers a physiologically relevant tool with which to characterize the molecular basis for interactions between Bordetella and its primary site of infection, the ciliated respiratory epithelium.

The presence of bacteriophages encoding Shiga toxin 2 (stx 2 phages) was analysed in 168 strains of Shiga-toxin-producing Escherichia coli (STEC) isolated from cattle. Following mitomycin C induction, strains carrying stx 2 phages were screened by plaque blot and hybridization with an stx 2 A-probe. In the stx 2-phage-carrying strains, the amounts of phage production, phage DNA extracted and Stx2 produced after induction were assessed. The induced stx 2 phages were characterized morphologically and genetically. Assays to obtain lysogens from different strains were also carried out and phages induced from the lysogens were compared with those induced from the STEC isolates. Results indicated that 18 % of the strains carried an inducible stx 2 phage. Most of them showed a direct relationship between phage induction and toxin production. Each strain carried only one inducible stx 2 phage, although a few strains had two copies of the stx 2 in the chromosome. The stx 2 phages showed diverse morphology and a wide variability in their genome. Assays to obtain lysogens showed that not all the phages were transduced with the same frequency and only six lysogens were obtained. Phages in the lysogens were the same as those induced from their respective initial STEC host strains, although the induction and relative toxin production of the lysogens varied. Most phages carried the stx 2 gene, while a few carried stx 2 variants. Infectivity of the phages depended on the different hosts, although O157 : H7 was preferentially infected by phages induced from O157 strains. The results show that inducible stx 2 phages are common among STEC of animal origin and that they may enhance the spread of stx 2.

To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.

In Neisseria meningitidis, the reversible expression of surface antigens, i.e. phase variation, results from changes within repeated simple sequence motifs located in coding or promoter regions of the genes involved in their biosynthesis. The mutation rates of these simple sequences, which have a major influence on the generation of phenotypic diversity, can affect the fitness of the population. The aim of the present study was to investigate the involvement of genetic factors involved (mutS and dam) and not yet analysed (drg and dinB) in the regulation of phase variation frequencies of genes associated with a variety of repeat tracts. The frequency of frameshifts occurring in the polycytidine (polyC) tracts associated with siaD, spr and lgtG and in the tetranucleotide (TAAA) repeat tract associated with nadA was determined by colony immunoblotting or using the lacZ gene as a reporter. Inactivation of mutS increased the frequency of phase variation of genes presenting homopolymeric tracts of diverse length. Overexpression of dinB enhanced the instability of the homopolymeric tract associated with siaD. Investigation of the dam locus in a population of genetically distinct N. meningitidis strains revealed that 27 % of strains associated with invasive disease contained the dam gene. In all strains where a Dam function was absent, the drg gene had been inserted into the dam locus. Disruption of dam and drg in strains representative of each genotype, i.e. dam +/drg and dam/drg +, did not modify phase variation frequencies. In contrast to the effects of certain genes on homopolymeric tracts, none of the genetic factors investigated affected the stability of tetranucleotide repeat tracts.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.