- Volume 161, Issue 3, 2015

The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of Mycobacterium tuberculosis, have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that espA is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates espA, but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the espR promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the espR start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of espR. Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for espR, espA and espD, and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.

Aspergillus fumigatus is considered to be the most prevalent airborne pathogenic fungus and can cause invasive diseases in immunocompromised patients. It is known that its virulence is multifactorial, although the mechanisms of pathogenicity remain unclear. With the aim of improving our understanding of these mechanisms, we designed a new expression microarray covering the entire genome of A. fumigatus. In this first study, we analysed the transcriptomes of this fungus at the first steps of germination after being grown at 24 and 37 °C. The microarray data revealed that 1249 genes were differentially expressed during growth at these two temperatures. According to our results, A. fumigatus modified significantly the expression of genes related to metabolism to adapt to new conditions. The high percentages of genes that encoded hypothetical or unclassified proteins differentially expressed implied that many as yet unknown genes were involved in the establishment of A. fumigatus infection. Furthermore, amongst the genes implicated in virulence upregulated at 37 °C on the microarray, we found those that encoded proteins mainly related to allergens (Asp F1, Asp F2 and MnSOD), gliotoxin biosynthesis (GliP and GliZ), nitrogen (NiiA and NiaD) or iron (HapX, SreA, SidD and SidC) metabolism. However, gene expression in iron and nitrogen metabolism might be influenced not only by heat shock, but also by the availability of nutrients in the medium, as shown by the addition of fresh medium.

Most colicins kill Escherichia coli cells by membrane pore formation or nuclease activity and, superficially, the mechanisms are similar: receptor binding, translocon recruitment, periplasmic receptor binding and membrane insertion. However, in detail, they employ a wide variety of molecular interactions that reveal a high degree of evolutionary diversification. Group A colicins bind to members of the TolQRAB complex in the periplasm and heterotrimeric complexes of colicin–TolA–TolB have been observed for both ColA and ColE9. ColN, the smallest and simplest pore-forming colicin, binds only to TolA and we show here that it uses the binding site normally used by TolB, effectively preventing formation of the larger complex used by other colicins. ColN binding to TolA was by β-strand addition with a K D of 1 µM compared with 40 µM for the TolA–TolB interaction. The β-strand addition and ColN activity could be abolished by single proline point mutations in TolA, which each removed one backbone hydrogen bond. By also blocking TolA–TolB binding these point mutations conferred a complete tol phenotype which destabilized the outer membrane, prevented both ColA and ColE9 activity, and abolished phage protein binding to TolA. These are the only point mutations known to have such pleiotropic effects and showed that the TolA–TolB β-strand addition is essential for Tol function. The formation of this simple binary ColN–TolA complex provided yet more evidence of a distinct translocation route for ColN and may help to explain the unique toxicity of its N-terminal domain.

The spirochaete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common tick-borne infection in the northern hemisphere. There is a long-standing debate regarding the role of pleomorphic forms in Lyme disease pathogenesis, while very little is known about the characteristics of these morphological variants. Here, we present a comprehensive analysis of B. burgdorferi pleomorphic formation in different culturing conditions at physiological temperature. Interestingly, human serum induced the bacterium to change its morphology to round bodies (RBs). In addition, biofilm-like colonies in suspension were found to be part of B. burgdorferi’s normal in vitro growth. Further studies provided evidence that spherical RBs had an intact and flexible cell envelope, demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the RBs displayed lower metabolic activity compared with spirochaetes. Furthermore, our results indicated that the different pleomorphic variants were distinguishable by having unique biochemical signatures. Consequently, pleomorphic B. burgdorferi should be taken into consideration as being clinically relevant and influence the development of novel diagnostics and treatment protocols.

Thioredoxin (Trx) maintains intracellular thiol groups in a reduced state and is involved in a wide range of cellular processes, including ribonucleotide reduction, sulphur assimilation, oxidative stress responses and arsenate detoxification. The industrially important lactic acid bacterium Lactococcus lactis contains two Trxs. TrxA is similar to the well-characterized Trx homologue from Escherichia coli and contains the common WCGPC active site motif, while TrxD is atypical and contains an aspartate residue in the active site (WCGDC). To elucidate the physiological roles of the two Trx paralogues, deletion mutants ΔtrxA, ΔtrxD and ΔtrxAΔtrxD were constructed. In general, the ΔtrxAΔtrxD strain was significantly more sensitive than either of the ΔtrxA and ΔtrxD mutants. Upon exposure to oxidative stress, growth of the ΔtrxA strain was diminished while that of the ΔtrxD mutant was similar to the wild-type. The lack of TrxA also appears to impair methionine sulphoxide reduction. Both ΔtrxA and ΔtrxD strains displayed growth inhibition after treatment with sodium arsenate and tellurite as compared with the wild-type, suggesting partially overlapping functions of TrxA and TrxD. Overall the phenotype of the ΔtrxA mutant matches established functions of WCGPC-type Trx while TrxD appears to play a more restricted role in stress resistance of Lac . lactis.

In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also known as candicidin, consists of 21 genes, including four regulatory genes, fscRI–fscRIV. Our bioinformatics analyses indicate that FscRI has an N-terminal PAS domain, whereas the other three regulators have N-terminal AAA domains and are members of the LAL (large ATP-binding regulators of the LuxR type) family. Deletion of fscRI abolished the production of FR-008, with production restored in the complemented strain, supporting a critical role for FscRI in FR-008 biosynthesis. Consistent with these findings, transcription of genes involved in the biosynthesis and efflux of FR-008 was greatly downregulated in a ΔfscRI mutant. Interestingly, the regulatory gene fscRIV was also downregulated in the ΔfscRI mutant. Production of FR-008 was reduced, but not abrogated, in an fscRIV deletion mutant, and although structural genes were downregulated in ΔfscRIV, the changes were much less dramatic than in ΔfscRI, suggesting a stronger regulatory role for FscRI. Remarkably, transcription of fscRI was also decreased in ΔfscRIV. Expression of fscRI restored antibiotic production in a ΔfscRIV mutant, but not vice versa. Putative binding sequences for FscRI were identified upstream of fscRIV and the three structural genes fscA, fscB and fscD, which encode large modular polyketide synthases. Our findings suggest that fscRI and fscRIV are interregulatory, whereas expression of fscRII and fscRIII appears to be independent of fscRI and fscRIV. This study demonstrates that the regulation of polyene antibiotic synthesis can involve mutually regulated transcriptional activators that belong to different families.

Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated β-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus’ re-entry into the cell cycle after termination of fatty acid starvation.

During a succession of phocine morbillivirus outbreaks spanning the past 25 years, Bordetella bronchiseptica was identified as a frequent secondary invader and cause of death. The goal of this study was to evaluate genetic diversity and the molecular basis for host specificity among seal isolates from these outbreaks. MLST and PvuII ribotyping of 54 isolates from Scottish, English or Danish coasts of the Atlantic or North Sea revealed a single, host-restricted genotype. A single, novel genotype, unique from that of the Atlantic and North Sea isolates, was found in isolates from an outbreak in the Caspian Sea. Phylogenetic analysis based either on MLST sequence, ribotype patterns or genome-wide SNPs consistently placed both seal-specific genotypes within the same major clade but indicates a distinct evolutionary history for each. An additional isolate from the intestinal tract of a seal on the south-west coast of England has a genotype otherwise found in rabbit, guinea pig and pig isolates. To investigate the molecular basis for host specificity, DNA and predicted protein sequences of virulence genes that mediate host interactions were used in comparisons between a North Sea isolate, a Caspian Sea isolate and each of their closest relatives as inferred from genome-wide SNP analysis. Despite their phylogenetic divergence, fewer nucleotide and amino acid substitutions were found in comparisons of the two seal isolates than in comparisons with closely related strains. These data indicate isolates of B. bronchiseptica associated with respiratory disease in seals comprise unique, host-adapted and highly clonal populations.

The phage P106B (vB_RglS_P106B) is a Siphoviridae phage with a narrow spectrum of infectivity, which has been isolated from soils with a history of pea cultivation. The trapping host of P106B is an indigenous strain of Rhizobium gallicum (SO14B-4) isolated from soils associated with Vicia cracca. Phenotypic characterization of the phage revealed that P106B has an approximate burst size of 21 p.f.u. per infected cell with 60 min and 100 min eclipse and latent periods, respectively. Phage P106B was unable to transduce under the conditions tested. The genome of P106B is 56 024 bp in length with a mean DNA G+C content of 47.9 %. The complete genome sequence contains 95 putative ORFs and a single tRNA gene coding for leucine with the anticodon TTA. Putative functions could only be assigned to 22 of the predicted ORFs while a significant number of ORFs (47) shared no sequence similarities to previously characterized proteins. The remaining 26 putative protein-coding genes exhibited a sequence resemblance to other hypothetical proteins. No lysogeny-related genes were found in the P106B genome.

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen–host interaction. The molecular mechanisms controlling and facilitating internalization of C or. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen–host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

The present study was conducted to determine the potential of five cyanobacteria strains isolated from aquatic zones to induce lipid production. The phylogenetic affiliation of the isolates was determined by 16S rRNA gene sequencing. Amongst the isolates, an efficient cyanobacterium, Synechococcus sp. HS01 showing maximal biomass and lipid productivity, was selected for further studies. In order to compare lipid productivity, the HS01 strain was grown in different media to screen potential significant culture ingredients and to evaluate mixotrophic cultivation. Mixotrophic cultivation of the strain using ostrich oil as a carbon source resulted in the best lipid productivity. GC analysis of fatty acid methyl esters of the selected cyanobacterial strain grown in media supplemented with ostrich oil showed a high content of C16 (palmitoleic acid and palmitic acid) and C18 (linoleic acid, oleic acid and linolenic acid) fatty acids of 42.7 and 42.8 %, respectively. Transmission electron micrographs showed that the HS01 cells exhibited an elongated rod-shaped appearance, either isolated, paired, linearly connected or in small clusters. According to initial experiments, ostrich oil, NaNO3 and NaCl were recognized as potential essential nutrients and selected for optimization of media with the goal of maximizing lipid productivity. A culture optimization technique using the response surface method demonstrated a maximum lipid productivity of 56.5 mg l−1 day−1. This value was 2.82-fold higher than that for the control, and was achieved in medium containing 1.12 g l−1 NaNO3, 1 % (v/v) ostrich oil and 0.09 % (w/v) NaCl.

S-Adenosyl-l-methionine (AdoMet) is an essential metabolite, serving in a very wide variety of metabolic reactions. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for antimicrobial agents. We previously showed that a variety of methionine analogues are MAT substrates, yielding AdoMet analogues that function in specific methyltransfer reactions. However, this left open the question of whether the modified AdoMet molecules could support bacterial growth, meaning that they functioned in the full range of essential AdoMet-dependent reactions. The answer matters both for insight into the functional flexibility of key metabolic enzymes, and for drug design strategies for both MAT inhibitors and selectively toxic MAT substrates. In this study, methionine analogues were converted in vitro into AdoMet analogues, and tested with an Escherichia coli strain lacking MAT (ΔmetK) but that produces a heterologous AdoMet transporter. Growth that yields viable, morphologically normal cells provides exceptionally robust evidence that the analogue functions in every essential reaction in which AdoMet participates. Overall, the S-adenosylated derivatives of all tested l-methionine analogues modified at the carboxyl moiety, and some others as well, showed in vivo functionality sufficient to allow good growth in both rich and minimal media, with high viability and morphological normality. As the analogues were chosen based on incompatibility with the reactions via which AdoMet is used to produce acylhomoserine lactones (AHLs) for quorum sensing, these results support the possibility of using this route to selectively interfere with AHL biosynthesis without inhibiting bacterial growth.