Stephen P. Kidd

The MerR family of transcriptional regulators

The MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognised. The signature of the family is amino acid similarity in the first 100 amino acids, including a helix-turn-helix motif followed by a coiled-coil region. With increasing recognition of members of this class over the last decade, particularly with the advent of rapid bacterial genome sequencing, MerR-like regulators have been found in a wide range of bacterial genera, but not yet in archaea or eukaryotes. The few MerR-like regulators that have been studied experimentally have been shown to activate suboptimal sigma(70)-dependent promoters, in which the spacing between the -35 and -10 elements recognised by the sigma factor is greater than the optimal 17+/-1 bp. Activation of transcription is through protein-dependent DNA distortion. The majority of regulators in the family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. A subgroup of the family activates transcription in response to metal ions. This subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region, but it is not yet clear how metal discrimination occurs. This subgroup of MerR family regulators includes MerR itself and may have evolved to generate a variety of specific metal-responsive regulators by fine-tuning the sites of metal recognition.

Defenses against Oxidative Stress in Neisseria gonorrhoeae: a System Tailored for a Challenging Environment

SUMMARY Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

A pneumococcal MerR-like regulator and S-nitrosoglutathione reductase are required for systemic virulence.

A transcriptional regulator, NmlR(sp), has been identified in Streptococcus pneumoniae that is required for defense against nitric oxide (NO) stress. The nmlR(sp) gene is cotranscribed with adhC, which encodes an alcohol dehydrogenase that is able to reduce S-nitrosoglutathione (GSNO) with NADH as reductant. nmlR(sp) and adhC mutants exhibited a reduced level of NADH-GSNO oxidoreductase activity and were more susceptible to killing by NO than were wild-type cells. Comparison of the virulence of wild-type and mutant strains by use of a mouse model system showed that NmlR(sp) and AdhC do not play a key role in the adherence of pneumococci to the nasopharynx in vivo. An intraperitoneal challenge experiment revealed that both NmlR(sp) and AdhC were required for survival in blood. These data identify novel components of a NO defense system in pneumococci that are required for systemic infection.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR‐like regulator (NmlR –Neisseria merR‐like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR‐like transcription factors with a distinctive  pattern  of  conserved  cysteines  within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione‐dependent dehydrogenase (AdhC), a CPx‐type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had >  50‐fold lower NADH‐S‐nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR‐like operator/promoter arrangements consisting of a dyad symmetry located between the −35 and −10 elements of the target genes. The NmlR target operator/promoters were cloned into a β‐galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress PAdhC (or PCopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)‐dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site‐directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild‐type. Analysis of regulation of MntC expression revealed that it was de‐repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn‐dependent peroxide‐responsive regulator found in Gram‐positive organisms. A perR mutant expressed more MntC protein than wild‐type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB‐dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Thioredoxin Reductase Is Essential for Protection of Neisseria gonorrhoeae against Killing by Nitric Oxide and for Bacterial Growth during Interaction with Cervical Epithelial Cells

In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.

Mercury Resistance Determinants Related to Tn21, Tn1696, and Tn5053 in Enterobacteria from the Preantibiotic Era

ABSTRACT Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion elements.

Glutathione-Dependent Alcohol Dehydrogenase AdhC Is Required for Defense against Nitrosative Stress in Haemophilus influenzae

ABSTRACT In Haemophilus influenzae Rd KW20, we identified a gene, adhC, which encodes a class III alcohol dehydrogenase (AdhC) and has S-nitrosoglutathione reductase activity. adhC exists on an operon with estD, which encodes an esterase. Divergent to the adhC-estD operon is the Haemophilus influenzae nmlR gene (nmlRHI), which encodes a MerR family regulator that is homologous to the Neisseria MerR-like regulator (NmlR). Analysis of an nmlRHI mutant indicated that expression of the adhC-estD operon is regulated by NmlRHI in strain Rd KW20. Chromosomal inactivation of either adhC or nmlRHI resulted in sensitivity to S-nitrosoglutathione and decreased S-nitrosoglutathione reductase activity. Examination of the NmlRHI-AdhC system in the genome sequences of nontypeable H. influenzae strains R2846, R2866, and 86-028NP identified significant variations. The adhC gene of 86-028NP was predicted to be nonfunctional due to a premature stop codon. Polymorphisms in the operator/promoter region of R2866 resulted in reduced enzyme activity. This correlated with an increased sensitivity to S-nitrosoglutathione. The adhC-nmlRHI system was examined in thirty-three clinical isolates (both capsular and nontypeable strains). Nucleic acid sequence data showed that only strain 86-028NP contained a premature stop codon. There were some variations in the DNA sequence of the operator/promoter region which altered the nmlRHI promoter. However, the clinical isolates still possessed S-nitrosoglutathione reductase activity and showed at least the equivalent ability to grow in the presence of S-nitrosoglutathione as Rd KW20. These data suggest that the nmlRHI-adhC system has a role in the defense against nitrosative stress in Haemophilus influenzae.

Phenotypic Characterization of a copA Mutant of Neisseria gonorrhoeae Identifies a Link between Copper and Nitrosative Stress

ABSTRACT NGO0579 is annotated copA in the Neisseria gonorrhoeae chromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, a copA mutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. The copA mutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, the copA mutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system of Neisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species.

Haemophilus influenzae and Streptococcus pneumoniae: living together in a biofilm

Streptococcus pneumoniae and Haemophilus influenzae are both commensals of the human nasopharynx with an ability to migrate to other niches within the human body to cause various diseases of the upper respiratory tract such as pneumonia, otitis media and bronchitis. They have long been detected together in a multispecies biofilm in infected tissue. However, an understanding of their interplay is a recent field of study, and while over recent years, there has been research that has identified many specific elements important in these biofilms, to date, it remains questionable whether the relationship between H. influenzae and S. pneumoniae is competitive or cooperative. Additionally, the factors that govern the nature of the interspecies interaction are still undefined. This review aims to collate the information that has emerged on the cocolonization and co-infection by S. pneumoniae and nontypeable H. influenzae (NTHi) and their formation of a multispecies biofilm.