Many bacteria encode biosynthetic proteins that produce a vast array of natural products. These compounds are often synthesized during host invasion as they function as virulence factors. In addition, such secondary metabolites have yielded numerous molecular scaffolds with pharmaceutical and clinical importance. The gene clusters that encode proteins responsible for synthesis of these compounds are typically silenced or “cryptic” under laboratory growth conditions, hampering discovery of novel lead compounds. We report here that MftR is a global repressor of secondary metabolite synthesis in Burkholderia thailandensis and that urate functions as a physiologically relevant inducer of gene expression. Biosynthetic gene clusters under MftR control include those associated with production of the antimicrobial bactobolins, the iron siderophore malleobactin, and the virulence factor malleilactone. MftR also controls additional genes associated with survival in a host environment, such as genes encoding components of the type III secretion system (T3SS) and proteins linked to anaerobic respiration. This observation not only has implications for understanding activation of gene regulatory networks during host invasion, but it also paves the way for isolation of novel therapeutic leads.

Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control.

SUMMARY Species within the genus Burkholderia exhibit remarkable phenotypic diversity. Genomic plasticity, including genome reduction and horizontal gene transfer, has been correlated with virulence traits in several species. However, the conservation of virulence genes in species otherwise considered to have limited potential for infection suggests that phenotypic diversity may not be explained solely on the basis of genetic diversity. Instead, differential organization and control of gene regulatory networks may underlie many phenotypic differences. In this review, we evaluate how regulation of gene expression by members of the multiple antibiotic resistance regulator (MarR) family of transcription factors may contribute to shaping the physiological diversity of Burkholderia species, with a focus on the clinically relevant human pathogens. All Burkholderia species encode a relatively large number of MarR proteins, a feature common to bacteria that must respond to environmental changes such as those associated with host invasion. However, evolution of gene regulatory networks has likely resulted in orthologous transcription factors controlling disparate sets of genes. Adaptation to, and survival in, diverse habitats, including a human or plant host, is key to the success of Burkholderia species as (opportunistic) pathogens, and recent reports suggest that control of virulence-associated genes by MarR proteins features prominently among the survival strategies employed by these species. We suggest that identification of MarR regulons will contribute significantly to clarification of virulence determinants and phenotypic diversity.

Second wave of COVID 19 pandemic in India came with unexpected quick speed and intensity, creating an acute shortage of beds, ventilators, and oxygen at the peak of occurrence. This may have been partly caused by emergence of new variant delta. Clinical experience with the cases admitted to hospitals suggested that it is not merely a steep rise in cases but also possibly the case profile is different. This study was taken up to investigate the differentials in the characteristics of the cases admitted in the second wave versus those admitted in the first wave. Records of a total of 14398 cases admitted in the first wave (2020) to our network of hospitals in north India and 5454 cases admitted in the second wave (2021) were retrieved, making it the largest study of this kind in India. Their demographic profile, clinical features, management, and outcome was studied. Age sex distribution of the cases in the second wave was not much different from those admitted in the first wave but the patients with comorbidities and those with greater severity had larger share. Level of inflammatory markers was more adverse. More patients needed oxygen and invasive ventilation. ICU admission rate remained nearly the same. On the positive side, readmissions were lower, and the duration of hospitalization was slightly less. Usage of drugs like remdesivir and IVIG was higher while that of favipiravir and tocilizumab was lower. Steroid and anticoagulant use remained high and almost same during the two waves. More patients had secondary bacterial and fungal infections in Wave 2. Mortality increased by almost 40% in Wave 2, particularly in the younger patients of age less than 45 years. Higher mortality was observed in those admitted in wards, ICU, with or without ventilator support and those who received convalescent plasma. No significant demographic differences in the cases in these two waves, indicates the role of other factors such as delta variant and late admissions in higher severity and more deaths. Comorbidity and higher secondary bacterial and fungal infections may have contributed to increased mortality.

In yeast, Hmo1p is important for communicating target of rapamycin (TOR) kinase activity to downstream targets. Results show that TOR kinase controls expression of the HMO1 gene and that an important component of this regulation is its direct association with the HMO1 gene. The implications are that TOR kinase may have more elaborate nuclear functions.

Our findings suggest that there is a definite increase in the multidrug resistant organisms. The data on the changing trends in antibiotic resistance, we believe is an important pillar in our efforts at improving infection control practices.

Biofilm formation by pathogenic Burkholderia species is a serious complication as it renders the bacteria resistant to antibiotics and host defenses. Using B. thailandensis, we report here a novel redox-sensitive member of the multiple antibiotic resistance regulator (MarR) protein family, BifR, which represses biofilm formation. BifR is encoded as part of the emrB-bif R operon; emrB-bif R is divergent to ecsC, which encodes a putative LasA protease. In Pseudomonas aeruginosa, LasA has been implicated in virulence by contributing to cleavage of elastase. BifR repressed the expression of ecsC and emrB-bif R, and expression was further repressed under oxidizing conditions. BifR bound two sites in the intergenic region between ecsC and emrB-bif R with nanomolar affinity under both reducing and oxidizing conditions; however, oxidized BifR formed a disulfide-linked dimer-of-dimers, a covalent linkage that was absent in BifR-C104A in which the redox-active cysteine was replaced with alanine. BifR also repressed an operon encoding enzymes required for synthesis of phenazine antibiotics, which function as alternate respiratory electron receptors, and inactivation of bif R resulted in enhanced biofilm formation. Taken together, our data suggest that BifR functions to control LasA production and expression of genes involved in biofilm formation, in part by regulating synthesis of alternate electron acceptors that promote survival in the oxygen-limiting environment of a biofilm. The correlation between increased repression of emrB-bif R under oxidative conditions and the formation of a covalently linked BifR dimer-of-dimers suggests that BifR may modulate gene activity in response to cellular redox state.

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