- Volume 154, Issue 8, 2008

Pseudomonas aeruginosa is a ubiquitous bacterium that causes opportunistic infections in a range of host tissues and organs. Infections by P. aeruginosa are difficult to treat and hence there is interest in the development of effective therapeutics. One of the key mechanisms that P. aeruginosa uses to control the expression of many virulence factors is the N-acylated homoserine lactone (AHL) regulatory system. Hence, there is considerable interest in targeting this regulatory pathway to develop novel therapeutics for infection control. P. aeruginosa is the principal cause of microbial keratitis and of infections in cystic fibrosis (CF) sufferers, and AHL-dependent cell-to-cell signalling has been shown to be important for both infection types. However, keratitis tends to be an acute infection whereas infection of CF patients develops into a chronic, life-long infection. Thus, it is unclear whether AHL-regulated virulence plays the same role during these infections. This review presents a comparison of the role of AHL signalling in P. aeruginosa-mediated microbial keratitis and chronic lung infections of CF patients.

The major cell wall carbohydrate of Corynebacterineae is arabinogalactan (AG), a branched polysaccharide that is essential for the physiology of these bacteria. Decaprenylphosphoryl-d-arabinose (DPA), the lipid donor of d-arabinofuranosyl residues of AG, is synthesized through a series of unique biosynthetic steps, the last one being the epimerization of decaprenylphosphoryl-β-d-ribose (DPR) into DPA, which is believed to proceed via a sequential oxidation–reduction mechanism. Two proteins from Mycobacterium tuberculosis (Rv3790 and Rv3791) have been shown to catalyse this epimerization in an in vitro system. The present study addressed the exact function of these proteins through the inactivation of the corresponding orthologues in Corynebacterium glutamicum (NCgl0187 and NCgl0186, respectively) and the analysis of their in vivo effects on AG biosynthesis. We showed that NCgl0187 is essential, whereas NCgl0186 is not. Deletion of NCgl0186 led to a mutant possessing an AG that contained half the arabinose and rhamnose, and less corynomycolates linked to AG but more trehalose mycolates, compared with the parental strain. A candidate gene that may encode a protein functionally similar to NCgl0186 was identified in both C. glutamicum (NCgl1429) and M. tuberculosis (Rv2073c). While the deletion of NCgl1429 had no effect on AG biosynthesis of the mutant, the gene could complement the mycolate defect of the AG of the NCgl0186 mutant, strongly supporting the concept that the two proteins play a similar function in vivo. Consistent with this, the NCgl1429 gene appeared to be essential in the NCgl0186-inactivated mutant. A detailed bioinformatics analysis showed that NCgl1429, NCgl0186, Rv3791 and Rv2073c could constitute, with 52 other proteins belonging to the actinomycetales, a group of closely related short-chain reductases/dehydrogenases (SDRs) with atypical motifs. We propose that the epimerization of DPR to DPA involves three enzymes that catalyse two distinct steps, each being essential for the viability of the bacterial cells.

The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc2155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant. RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88–99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein–protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Under conditions of nitrogen limitation, the general transcription factor TnrA in Bacillus subtilis activates the expression of genes involved in assimilation of various nitrogen sources. Previously, TnrA activity has been shown to be controlled by protein–protein interaction with glutamine synthetase, the key enzyme of ammonia assimilation. Furthermore, depending on ATP and 2-oxoglutarate levels, TnrA can bind to the GlnK–AmtB complex. Here, we report that upon transfer of nitrate-grown cells to combined nitrogen-depleted medium, TnrA is rapidly eliminated from the cells by proteolysis. As long as TnrA is membrane-bound through GlnK–AmtB interaction it seems to be protected from degradation. Upon removal of nitrogen sources, the localization of TnrA becomes cytosolic and degradation occurs. The proteolytic activity against TnrA was detected in the cytosolic fraction but not in the membrane, and its presence does not depend on the nitrogen regime of cell growth. The proteolytic degradation of TnrA as a response to complete nitrogen starvation might represent a novel mechanism of TnrA control in B. subtilis.

The transport of inorganic phosphate (Pi) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity Pi transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA–PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.

The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is used for degradation of natural, persistent aromatic polymers (lignin, humic acids and brown coal components). The N. frowardii MNP has shown good activity in conversion of xenobiotic compounds such as polycyclic hydrocarbons and trinitrotoluene. However, this biotechnologically promising fungus has not previously been studied at the molecular biology level. We show here that according to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3-, three lignin peroxidase- and two laccase-encoding genes, and the gene encoding the ribosomal SSU 18S RNA, that the fungus has a close phylogenetic relationship to the white-rot basidiomycete Phlebia radiata (Fr.). Ribosomal internal transcribed spacer (ITS) sequence (ITS1+5.8S+ITS2) phylogeny reclassifies Nf b19 as a possible representative of a new species of the genus Phlebia, nearest to the Phlebia acerina clade. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified in the order Agaricales, family Strophariaceae. Our results thus indicate a need for systematic re-identification of the previously named N. frowardii isolate b19.

In order to contribute to our understanding of Acidithiobacillus taxonomy, we determined 16S rDNA sequences and the 16S–23S internally transcribed spacer (ITS) sequence of 35 Chinese Acidithiobacillus isolates and three reference strains representing three validly described species and used them to construct phylogenetic trees. The two phylogenetic trees were roughly similar topologically, and Acidithiobacillus strains were assigned to eight phylogenetic groups. In addition, the results of phylogenetic analysis were consistent with those obtained by randomly amplified polymorphic DNA (RAPD) cluster analysis. Compared with a phylogenetic tree based on the 16S rRNA sequences, the ITS tree showed more clearly the inter- and intraspecific genealogical relationships of the genus Acidithiobacillus. Similarity values of the ITSs varied from 60.5 % to 84.7 % between representative strains of different species, and the maximum level of ITS divergence between strains belonging to the same species was 13 %. Coupling phylogenetic analysis and phenotypic characteristics, we concluded that at least each of the three Acidithiobacillus ferrooxidans phylogenetic groups should be considered a separate subspecies, and that five sulfur-oxidizing Chinese Acidithiobacillus-like isolates represent one or two new species of the genus Acidithiobacillus. The ITS may be a potential target for the development of fluorescent in situ hybridization probes for more accurately detecting distinct ecotypes of Acidithiobacillus strains and other closely related sulfur-oxidizing bacteria.

The genus Clostridium comprises a heterogeneous group of organisms for which the phylogeny and evolutionary relationships are poorly understood. The elucidation of these evolutionary relationships necessitates the use of experimental methods that can distinguish Clostridium lineages that are time and cost effective, and can be accurately and reproducibly employed in different laboratories. Multi-locus sequence typing (MLST) has been successfully used as a reproducible and discriminating system in the study of eukaryotic and prokaryotic evolutionary biology, and for strain typing of various bacteria. In this study, MLST was applied to evaluate the evolutionary lineages in the serotype A group of Clostridium botulinum. C. botulinum type A has recently been shown to produce multiple subtypes, suggesting that it is not monophyletic as previously reported, but comprises distinct lineages. For MLST analysis, we initially evaluated 14 housekeeping genes (gapdh, tuf, sod, oppB, hsp60, dnaE, aroE, pta, 23S rDNA, aceK, rpoB, 16S rDNA, mdh and recA) for amplification and sequence analysis. In the first phase of the analysis, 30 C. botulinum type A strains producing botulinum neurotoxin subtypes A1–A4 were examined. Results of this pilot study suggested that seven of the genes (mdh, aceK, rpoB, aroE, hsp60, oppB and recA) could be used for elucidation of evolutionary lineages and strain typing. These seven housekeeping genes were successfully applied for the elucidation of lineages for 73 C. botulinum type A strains, which resulted in 24 distinct sequence types. This strategy should be applicable to phylogenetic studies and typing of other C. botulinum serotypes and Clostridium species.

Exploring the evolutionary response of Bacillus simplex strains to the slope-specific habitats of ‘Evolution Canyon’ I and II, Israel, we report here on presumably adaptive differences in fatty acid (FA) content that correlate with one particular feature of the habitats, temperature difference. These two canyons represent similar ecological sites, separated by 40 km, in which the orientation of the sun yields a strong sun-exposed and hot ‘African’ south-facing slope versus a rather cooler and mesic-lush ‘European’ north-facing slope within a distance of only 50–400 m. Among 131 strains, which are identical in their 16S sequences, those assigned genetically to the ‘African’ ecotypes express phenotypically generally more high-temperature-tolerance-providing iso-branched FAs than strains assigned to the ‘European’ ecotypes when grown at 20 °C, 28 °C and 40 °C. Conversely, ‘European’ lineages express larger amounts of low-temperature-tolerance-providing anteiso-branched and non-saturated FAs when grown at the same temperatures. Moreover, ‘African’ ecotypes show a stronger adjustment of their high- and low-temperature-tolerance-providing FAs in response to low temperatures, which suggests that, as a result of temperature adaptation, ‘African’ and ‘European’ ecotypes have evolved different reaction norms within their phenotypic plasticity response. Thus, bacterial adaptive microevolution may include such multigenic and highly complex organs as the bacterial cell membrane. The results contribute to our understanding of the speciation process among the ‘Evolution Canyon’ B. simplex ecotypes.

During natural transformation of Acinetobacter baylyi, the genomic integration of foreign (non-homologous) DNA is possible when the DNA contains a single segment homologous to the recipient genome (anchor) through homologous recombination in the anchor facilitating illegitimate recombination in the neighbouring foreign DNA (homology-facilitated illegitimate recombination; HFIR). DNA integration by HFIR occurs about 10 000 times less frequently than fully homologous recombination, but at least 100 000-fold more frequently than integration in the absence of any homology. We investigated the influence of the RecBCD enzyme (DNase/helicase) and SbcCD DNase (DNA-structure-specific single-strand endonuclease and exonuclease) on HFIR. In a recBCD null mutant the acquisition of foreign DNA was elevated 11-fold relative to wild-type cells by a 6.9-fold increased HFIR frequency and by the integration of longer stretches of foreign DNA in each event. In an sbcCD null mutant, the foreign DNA acquisition was 4.5-fold higher than in the wild-type, while homologous transformation with large DNA molecules was unaffected and increased 3.2-fold with small DNA fragments. The sbcCD mutation partially suppressed the high UV sensitivity and low viability of the recBCD mutant and also decreased its foreign DNA acquisition by HFIR to the lower level of the sbcCD mutant. We propose that suppression of HFIR results from the elimination of double-stranded intermediates of the HFIR process during transformation by RecBCD, and by SbcCD interfering with branched molecules. Our results provide evidence that the homologous recombination enzymes RecBCD and SbcCD control the level of foreign DNA acquisition by HFIR.

Candida albicans, clinically the most important human fungal pathogen, rapidly develops resistance to antifungal drugs. The acquisition of resistance has been linked to various types of genome changes. As part of an ongoing study of this problem, we investigated mutation, genome stability and drug resistance acquisition in C. albicans strains with deletions in the base excision repair (BER) genes NTG1, APN1 and OGG1, and in the nucleotide excision repair (NER) genes RAD2 and RAD10. The BER mutants did not exhibit any change in their susceptibility to DNA-damaging agents, but the NER mutants were extremely sensitive to UV-induced DNA damage. We did not observe any significant change in mutation, genome stability and antifungal drug sensitivity in the mutant strains we tested. However, we detected a number of intriguing phenotypic differences between strains bearing deletions in equivalent C. albicans and Saccharomyces cerevisiae BER and NER genes, which may be related to differences in the life cycles of these two fungi.

Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases (EC 3.5.1.24) is of importance, especially for lactobacilli, which are considered to play a major role in bile salt hydrolysis in vivo. In the present study, all enzymes listed in public databases that belong to the Bsh family and the closely related penicillin V acylase (Pva; EC 3.5.1.11) family were compared with the sequences annotated as Bsh in Lactobacillus plantarum WCFS1, as an example. In Gram-positive bacteria, a clear distinction was made between the two families using sequence alignment, phylogenetic clustering, and protein homology modelling. Biochemical and structural data on experimentally verified Bsh and Pva enzymes were used for validation of function prediction. Hidden Markov models were constructed from the sequence alignments to enable a more accurate prediction of Bsh-encoding genes, and their distinction from those encoding members of the Pva family. Many Pva-related sequences appeared to be annotated incorrectly as Bsh in public databases. This refinement in the annotation of Bsh family members influences the prediction of the function of bsh-like genes in species of the genus Lactobacillus, and it is discussed in detail.

The current model for virulence gene regulation in Bacillus anthracis involves several trans-acting factors, the most important of which appears to be the anthrax toxin activator encoded by the atxA gene. AtxA is a positive regulator of the toxin genes pagA, cya and lef, and of a number of other plasmid- and chromosome-encoded genes. The AtxA protein (56 kDa) possesses a predicted winged-helix DNA-binding domain and phosphotransferase system-regulated domains, but the mechanism for positive regulation of AtxA target genes is not known. Sequence similarities in the promoter regions of AtxA-regulated genes are not apparent, and recombinant AtxA binds DNA with a high affinity in a non-specific manner. We hypothesized that the toxin genes possess common structural features or cis-acting elements that are required for positive regulation. We employed deletion analyses to determine the minimal sequences required for atxA-mediated toxin gene expression. In silico modelling and in vitro experiments using double-stranded DNA corresponding to the toxin gene promoter regions indicated significant curvature associated with these regions. These findings suggest that the structural topology of the DNA plays an important role in the control of anthrax toxin gene expression.

The dipeptide transport operon in bacteria comprises genes for the transport and metabolism of amino acids and dipeptides, as well as haem and haem precursors such as aminolaevulinic acid. Such nutrient and mineral sources are vital for bacteria to survive in and colonize a range of niches. In silico analysis of the dipeptide transport systems in sequenced Pseudomonas species identified the presence of two genes in P. aeruginosa strains that were absent in other sequenced pseudomonads. These genes encode a putative metallopeptidase, PA4498, and a putative transcriptional regulator, PA4499. Proteomic profiling of wild-type PAO1 and a PA4499 mutant strain indicated that PA4499 negatively regulated the putative peptidase, PA4498. Transcriptional fusion analysis verified that expression of PA4498 (mdpA, metallo-dipeptidase aeruginosa) was negatively regulated by the downstream putative transcriptional regulator PA4499 (psdR, Pseudomonas dipeptide regulator). Transcriptional fusion analysis also showed that the dppABCDF operon was under the negative control of psdR. Functional genomic analysis of mdpA indicated that it is required for the metabolism of a range of dipeptides and that it contributes to the cytotoxicity of PAO1 on an epithelial cell line.