- Volume 154, Issue 11, 2008

The dasABC genes encode an ATP-binding cassette (ABC) transporter, which is one of the uptake systems for N,N′-diacetylchitobiose [(GlcNAc)2] in Streptomyces coelicolor A3(2), although the gene encoding the ABC subunit that provides ATP hydrolysis for DasABC has not been identified. In this study, we disrupted the sequence that is highly homologous to the msiK gene, the product of which is an ABC subunit assisting several ABC permeases in other Streptomyces species. Disruption of msiK severely affected the ability of S. coelicolor A3(2) to utilize maltose, cellobiose, starch, cellulose, chitin and chitosan, but not glucose. The msiK null mutant lacked (GlcNAc)2-uptake activity, but GlcNAc transport activity was unaffected. The data indicated that msiK is essential for (GlcNAc)2 uptake, which in S. coelicolor A3(2) is governed by ABC transporters including the DasABC–MsiK system, in contrast to Escherichia coli and Serratia marcescens, in which (GlcNAc)2 uptake is mediated by the phosphotransferase system. Interestingly, the induction of chitinase production by (GlcNAc)2 or chitin was absent in the msiK null mutant, unlike in the parent strain M145. The defect in chitinase gene induction was rescued by expressing the His-tagged MsiK protein under the control of the putative native promoter on a multicopy plasmid. The data suggest that uptake of (GlcNAc)2 is necessary for induction of chitinase production. The msiK gene was constitutively transcribed, whereas the transcription of dasA [(GlcNAc)2-binding protein gene], malE (putative maltose-binding protein gene), cebE1 (putative cellobiose-binding protein gene) and bxlE1 (putative xylobiose-binding protein gene) was induced by their corresponding sugar ligands. This is believed to be the first report to indicate that (GlcNAc)2 uptake mediated by ABC transporters is essential for chitinase production in streptomycetes, which are known to be the main degraders of chitin in soil.

The DnaA protein is the bacterial initiator of replication at a unique chromosomal site, oriC. It is present in all bacterial species and has a conserved structure with four domains. The structures of domains I and III–IV have been solved recently for some bacterial species, and the molecular process leading to the initiation event has been investigated in detail. On the other hand, domain II appears to have no rigid structure and is assumed to be a flexible linker connecting the N-terminal domain I and the C-terminal domains III–IV. It differs significantly in length and amino acid sequence among bacterial species. Whether or not domain II has any function(s) to initiate replication is unknown. The precise borders at both of its ends as well as its essential portions for cell viability are also unknown. In this study, we introduced systematic deletions into the domain II region on the chromosomal dnaA gene of Escherichia coli and examined their effect on cell physiology. Stretches of 30–36 consecutive amino acid residues could be deleted from various portions between the 78th and the 136th residues without affecting cell viability. We propose that domain II of E. coli DnaA is from the 79th to the 135th residues and at least 21–27 residues are required as a spacer to keep domains I and III–IV in the correct positions.

Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO2 and C2H2O2 groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. jejuni.

Acinetobacter baumannii forms biofilms on abiotic surfaces, a phenotype that may explain its ability to survive in nosocomial environments and to cause device-related infections in compromised patients. The biofilm proficiency of the 19606 type strain depends on the production of pili, cell-surface appendages assembled via the CsuAB-A-B-C-D-E chaperone–usher secretion system. The screening of a bank of isogenic insertion derivatives led to the identification of a biofilm-deficient derivative in which a transposon insertion disrupted a gene predicted to encode the response regulator of a two-component regulatory system. This gene, which was named bfmR, is required for the expression of the Csu pili chaperone–usher assembly system. This coding region is followed by an ORF encoding a putative sensor kinase that was named bfmS, which plays a less relevant role in biofilm formation when cells are cultured in rich medium. Further examination showed that the bfmR mutant was capable of attaching to abiotic surfaces, although to levels significantly lower than those of the parental strain, when it was cultured in a chemically defined minimal medium. Additionally, the morphology of planktonic cells of this mutant, when grown in minimal medium, was drastically affected, while adherent mutant cells were indistinguishable in shape and size from the parental strain. Together, these results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.

Certain amino acids, and cysteine in particular, promptly blocked toxin expression in Clostridium difficile strain VPI 10463 when added to late-exponential-phase peptone-yeast cultures, i.e. prior to normal induction of toxins A and B. Glucose reduced toxin yields by 80-fold, but only when supplemented at inoculation. Forty upregulated C. difficile proteins were identified during maximum toxin expression, and most of these were enzymes involved in energy exchange, e.g. succinate, CO/folate and butyrate metabolism. Transcription of tcdA (toxin operon) and folD (CO/folate operon) was induced by 20- and 10-fold, respectively, and with strikingly similar kinetics between OD 0.8 and 1.2. The sigma factors tcdR and sigH were upregulated simultaneously with tcdA and folD (3.5-fold increase of mRNA level), whereas transcription of tcdC, codY, sigB and sigL showed little or no correlation with that of tcdA and folD. The results suggest a connection between toxin expression, alternative energy metabolism and initial sporulation events in C. difficile.

Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [γ-32P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

The double strand-specific endoRNase RNase III globally regulates the production of antibiotics by Streptomyces coelicolor. We have undertaken studies to determine whether the endoRNase activity of S. coelicolor RNase III or its RNA binding activity is responsible for its regulatory function. We show that an rnc null mutant of S. coelicolor M145 does not produce actinorhodin or undecylprodigiosin. Restoring a wild-type copy of rnc to that mutant also restored antibiotic production. We constructed an rnc point mutant, D70A, in which an aspartic acid residue which is essential for the catalytic activity of RNase III was changed to alanine. The D70A mutation abolished the catalytic activity of the protein but not its ability to bind to RNA substrates. Introduction of a copy of the D70A gene into the rnc null mutant did not restore antibiotic production. This result suggests that the endoRNase activity of RNase III is required for the regulation of antibiotic production in S. coelicolor. We also reconstructed the C120 point mutation that was originally described in 1992. Although that mutation diminished antibiotic production by S. coelicolor, we confirm here that the C120 protein retains some RNase III activity.

Elsinochromes are nonhost-selective, light-activated, polyketide-derived toxins produced by many phytopathogenic Elsinoë species. We recently showed that the polyketide synthase-encoding gene EfPKS1 is essential for elsinochrome biosynthesis in the citrus scab fungus Elsinoë fawcettii. Sequence analysis beyond the EfPKS1 gene identified nine putative ORFs: four genes, designated RDT1, TSF1, PRF1 and ECT1, all encode polypeptides likely to have biosynthetic or efflux functions; five additional genes, OXR1 and EfHP1 to EfHP4, encode hypothetical proteins of unknown function. Northern-blot analysis revealed that expression of these genes in E. fawcettii was not completely correlated with accumulation of elsinochromes under nitrogen limitation, alkaline pH or high concentrations of glucose. Targeted disruption of the TSF1 gene, encoding a putative transcriptional activator, yielded fungal mutants unable to produce elsinochromes, and defective in both conidiation and expression of RDT1, EfPKS1, PRF1 and EfHP1, whereas expression of RDT1, TSF1, PRF1 and ECT1 was nearly abolished in EfPKS1-disrupted mutants. By contrast, expression of OXR1, EfHP2 and EfHP3 was not affected by disrupting either EfPKS1 or TSF1. Taken together, the results indicate that in addition to polyketide synthase, the products of TSF1 and other adjacent genes may also play a crucial role in elsinochrome production.

The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells, resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrfA* mutant proteins exhibiting differing degrees of activity were purified and analysed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion, conformational changes were detected for the PrfA* mutant proteins in comparison to wild-type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay appeared to directly correlate with levels of PrfA mutational activation, such that the high-activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein–protein interactions.

The ectomycorrhizal fungus Thelephora ganbajun is an endemic gourmet mushroom in Yunnan province, south-western China. However, despite its widespread consumer appeal, nutritional value and potential ecological role in natural forests, very little is known about its genetics, diversity and ecology. In this study, we investigated DNA sequence variation at the internal transcribed spacer (ITS) regions among 156 specimens collected from 23 sites of nine regions in Yunnan Province. Our analysis identified a total of 34 ITS haplotypes and these haplotypes were clustered into five distinct phylogenetic groups. The evolutionary divergences among these clades are similar to or greater than many known sister species pairs within the genus Thelephora and the closely related genus Tomentella. Among the 34 ITS haplotypes, 22 were represented by one specimen each and the remaining 12 were each shared by two or more specimens. The most common haplotype contained 68 specimens distributed in 21 of the 23 sites, a result consistent with gene flow among geographical populations. However, analysis of molecular variance (AMOVA) revealed low but significant genetic differentiation among local and regional populations. Interestingly, the Mantel test identified that the extent of genetic differentiation was not significantly correlated with geographical distance. Our study revealed significant genetic divergence within Th. ganbajun and limited but detectable gene flow among geographical populations of this endemic ectomycorrhizal gourmet mushroom.

Degradation of butyl benzyl phthalate (BBP) by a co-culture of Gordonia sp. strain MTCC 4818 and Arthrobacter sp. strain WY was investigated. In the degradation of BBP by the co-culture, the limitations of the individual species in metabolizing BBP were overcome, leading to the development of a consortium capable of complete utilization of this ester. In the degradation of BBP by the co-culture, the presence of multiple esterases was demonstrated in both species by activity staining of non-denaturing gels, indicating their roles in the degradation process. The esterases were found to be inducible, with unique or broad substrate specificities towards BBP and its monoesters. Moreover, a number of catabolic enzymes other than esterases identified in the metabolism of BBP-degraded intermediates facilitated the co-culture-mediated degradation process. The versatility of the co-culture was further established by the rapid and complete degradation of a mixture of phthalate esters of environmental concern.

Phylogenetic relationships among picocyanobacteria from the Syn/Pro clade sensu Sánchez-Baracaldo et al. (2005) were determined using small subunit (ssu) rDNA sequences from novel culture isolates together with environmental samples from the Baltic Sea and seven freshwater lakes. The picocyanobacterial community comprised members of previously identified clades and of two previously undescribed clades. The number of well-supported clades suggests that freshwater picocyanobacterial communities encompass much greater diversity than is found in marine systems. To allow the quantification of community structure and temporal succession, clade-specific ssu rDNA TaqMan assays were designed and implemented. These assays were used to assess picocyanobacterial community structure in two lakes over an annual cycle in 2003/4, and in a small number of Baltic Sea samples collected in July 2003. In the lake-water samples, picocyanobacteria were found to be scarce during most of the year, with members of each clade reaching their peak abundance over a relatively short period during the summer (June to September), although representatives of the Cyanobium clade also developed an autumn peak extending towards the end of October. All four freshwater clades were present in the Baltic Sea, but their distribution was patchy over relatively short spatial scales. The use of molecular tools for describing and quantifying community structures reveals previously unexplored complexity in the phytoplankton and will facilitate the development of a more sophisticated understanding of community dynamics at the base of the food chains in lakes.

Optical maps for five representative clinical, food-borne and bovine-derived isolates from the 2006 Escherichia coli O157 : H7 outbreak linked to fresh spinach in the United States showed a common set of 14 distinct chromosomal markers that define the outbreak strain. Partial 454 DNA sequencing was used to characterize the optically mapped chromosomal markers. The markers included insertions, deletions, substitutions and a simple single nucleotide polymorphism creating a BamHI site. The Shiga toxin gene profile of the spinach-associated outbreak isolates (stx1− stx2 + stx2c +) correlated with prophage insertions different from those in the prototypical EDL933 and Sakai reference strains (stx1+ stx2 + stx2c −). The prophage occupying the yehV chromosomal position in the spinach-associated outbreak isolates was similar to the stx1+ EDL933 cryptic prophage V, but it lacked the stx1 gene. In EDL933, the stx2 genes are within prophage BP933-W at the wrbA chromosomal locus; this locus was unoccupied in the spinach outbreak isolates. Instead, the stx2 genes were found within a chimeric BP933-W-like prophage with a different integrase, inserted at the argW locus in the outbreak isolates. An extra set of Shiga toxin genes, stx2c, was found in the outbreak isolates within a prophage integrated at the sbcB locus. The optical maps of two additional clinical isolates from the outbreak showed a single, different prophage variation in each, suggesting that changes occurred in the source strain during the course of this widespread, multi-state outbreak.

SH1 is the only reported isolate of a spherical halovirus, a dominant morphotype in hypersaline lakes. The virus lytically infects the haloarchaeon Haloarcula hispanica, and carries a 30.9 kb linear dsDNA genome that, in a previous study, was proposed to contain 56 protein-coding genes, probably organized into between four and eight operons. In the present study, these predictions were directly tested by determining the orientations and lengths of virus transcripts using systematic RT-PCR and primer extension. Seven major transcripts were observed that together covered most of the genome. Six transcripts were synthesized from early in infection (1 h post-infection; p.i.) onwards, while transcript T6 was only detected late in infection (5–6 h p.i.). No transcripts were detected in the inverted terminal repeat sequences or at the extreme right end of the genome (ORFs 55–56). Start points for the major transcripts were mapped by primer extension and corresponded closely to the 5′ termini determined by RT-PCR. Between 1 and 4 h p.i., transcripts usually terminated not far beyond the end of their last coding ORF, but late in infection, transcripts from the same promoters often terminated at more distal points, resulting in much of the genome being transcribed from both strands. Since many of these transcripts are complementary, RNA–RNA interactions are likely, and may play a role in regulating viral gene expression. Puromycin blockage of post-infection protein synthesis significantly altered the levels of certain virus transcripts, indicating that de novo protein synthesis is essential for the correct regulation of SH1 gene expression.