- Volume 164, Issue 9, 2018

Lipid metabolism is critical to Mycobacterium tuberculosis survival and infection. Unlike Escherichia coli, which has a single FadR, the M. tuberculosis genome encodes five proteins of the FadR sub-family. While the role of E. coli FadR as a regulator of fatty acid metabolism is well known, the definitive functions of M. tuberculosis FadR proteins are still under investigation. An interesting question about the M. tuberculosis FadRs remains open: which one of these proteins is the functional homologue of E. coli FadR? To address this, we have applied two different approaches. The first one was the bioinformatics approach and the second one was the classical molecular genetic approach involving complementation studies. Surprisingly, the results of these two approaches did not agree. Among the five M. tuberculosis FadRs, Rv0494 shared the highest sequence similarity with FadR E. coli and Rv0586 was the second best match. However, only Rv0586, but not Rv0494, could complement E. coli ∆fadR, indicating that Rv0586 is the M. tuberculosis functional homologue of FadR E. coli . Further studies showed that both regulators, Rv0494 and Rv0586, show similar responsiveness to LCFA, and have conserved critical residues for DNA binding. However, analysis of the operator site indicated that the inter-palindromic distance required for DNA binding differs for the two regulators. The differences in the binding site selection helped in the success of Rv0586 binding to fadB upstream over Rv0494 and may have played a critical role in complementing E. coli ∆fadR. Further, for the first time, we report the lipid-responsive nature of Rv0586.

The synthesis of methionine is critical for most bacteria. It is known that cellular methionine has a feedback effect on the expression of met genes involved in de novo methionine biosynthesis. Previous studies revealed that Gram-negative bacteria control met gene expression at the transcriptional level by regulator proteins, while most Gram-positive bacteria regulate met genes at post-transcriptional level by RNA regulators (riboregulators) located in the 5′UTR of met genes. However, despite its importance, the methionine biosynthesis pathway in the Gram-negative Xanthomonas genus that includes many important plant pathogens is completely uncharacterized. Here, we address this issue using the crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc), a model bacterium in microbe–plant interaction studies. The work identified an operon (met) involved in de novo methionine biosynthesis in Xcc. Disruption of the operon resulted in defective growth in methionine-limited media and in planta. Western blot analysis revealed that the expression of the operon is dependent on methionine levels. Further molecular analyses demonstrated that the 5′UTR, but not the promoter of the operon, is involved in feedback regulation on operon expression in response to methionine availability, providing an example of a Gram-negative bacterium utilizing a 5′UTR region to control the expression of the genes involved in methionine biosynthesis.

Burkholderia pseudomallei, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The bpeE-bpeF-oprC genes are distally located in the llpE-bpeE-bpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE′-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpE-bpeE-bpeF-oprC operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N₁₁-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli.

Mycobacteriophage D29 is a lytic phage that infects various species of Mycobacterium including M. tuberculosis. Its genome has 77 genes distributed almost evenly between two converging operons designated as left and right. Transcription of the phage genome is negatively regulated by multiple copies of an operator-like element known as stoperator that acts by binding the phage repressor Gp71. The function of the D29 genes and their expression status are poorly understood and therefore we undertook a transcriptome analysis approach to address these issues. The results indicate that the average transcript intensity of the right arm genes was higher than of those on the left, at the early stage of infection. Moreover, the fold increase from early to the late stage was found to be less for the right arm genes than for the left. Both observations support the prediction that the right arm genes are expressed early whereas the left arm ones are expressed late. The analysis further revealed a break in the continuity of the right arm operon between 89, the first gene in it, and 88, the next. Gene 88 was found to be expressed from a newly identified promoter located between 88 and 89. Another new promoter was found upstream of 89. Thus, the promoter P_left, identified earlier, is not the only one that drives expression of the right arm genes. All these promoters overlap with stoperators, with which they share a conserved sequence motif, TTGACA, commonly known as the −35 promoter element. We demonstrate mutually exclusive binding of RNA polymerase and Gp71 to the stoperator-promoters and conclude that stoperators can function as −35 promoter elements and that they can control gene expression not only negatively as was believed earlier but in many cases positively as well.

A monooxygenase-encoding gene (Mono) is located in the hypocrellin gene cluster of Shiraia sp. SUPER-H168 and was targeted by a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. The ΔMono mutant abolished hypocrellin production, whereas the ΔMono complement mutant restored hypocrellin production. Relative expression levels of the Mono and its adjacent genes were abolished in the ΔMono mutant compared with the wild-type strain. These results indicate the essential role of Mono in hypocrellin biosynthesis. The Mono gene of Shiraia bambusicola was further expressed in Pichia pastoris and salicylate monooxygenase activity was detected, which suggested that this monooxygenase has the ability to catalyse decarboxylative hydroxylation. The relative growth ratio of the ΔMono mutant was significantly improved compared with the wild-type strain. In contrast to the wild-type strain, the ΔMono mutant also represented excellent oxidative stress tolerance after exposure to high concentrations of H₂O₂ (16 mM) based on the increasing activities of superoxide dismutase, catalase, and glutathione peroxidase. These results suggest that ΔMono mutants could be used as microbial cell factories to produce metabolites that will cause oxidative stress. This study also enhances our understanding of hypocrellin biosynthesis and opens an avenue for decoding the hypocrellin pathway.

Nasal colonization by the pathogen Staphylococcus aureus is a risk factor for subsequent infection. Loss of function mutations in the gene encoding the virulence regulator Rsp are associated with the transition of S. aureus from a colonizing isolate to one that causes bacteraemia. Here, we report the identification of several novel activity-altering mutations in rsp detected in clinical isolates, including for the first time, mutations that enhance agr operon activity. We assessed how these mutations affected infection-relevant phenotypes and found loss and enhancement of function mutations to have contrasting effects on S. aureus survival in blood and antibiotic susceptibility. These findings add to the growing body of evidence that suggests S. aureus ‘trades off’ virulence for the acquisition of traits that benefit survival in the host, and indicates that infection severity and treatment options can be significantly affected by mutations in the virulence regulator rsp.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2–lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6–lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2–lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.