Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon

ABSTRACT The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE , previously annotated gdhR in Neisseria meningitidis , is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA , which encodes an l -glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae , expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA , as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection. IMPORTANCE The pathogenic Neisseria species are strict human pathogens that can cause a sexually transmitted infection ( N. gonorrhoeae ) or meningitis or fulminant septicemia ( N. meningitidis ). Although they share considerable genetic information, little attention has been directed to comparing transcriptional regulatory systems that modulate expression of their conserved genes. We hypothesized that transcriptional regulatory differences exist between these two pathogens, and we used the gdh locus as a model to test this idea. For this purpose, we studied two conserved genes ( gdhR and gdhA ) within the locus. Despite general conservation of the gdh locus in gonococci and meningococci, differences exist in noncoding sequences that correspond to promoter elements or potential sites for interacting with DNA-binding proteins, such as GdhR and MtrR. Our results indicate that implications drawn from studying regulation of conserved genes in one pathogen are not necessarily translatable to a genetically related pathogen

Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins

The definition of a typical sec-dependent bacterial signal peptide contains a positive charge at the N-terminus, thought to be required for membrane association. In this study the amino acid distribution of all Escherichia coli secretory proteins were analysed. This revealed that there was a statistically significant bias for lysine at the second codon position (P2), consistent with a role for the positive charge in secretion. Removal of the positively charged residue P2 in two different model systems revealed that a positive charge is not required for protein export. A well-characterized feature of large amino acids like lysine at P2 is inhibition of N-terminal methionine removal by methionyl amino-peptidase (MAP). Substitution of lysine at P2 for other large or small amino acids did not affect protein export. Analysis of codon usage revealed that there was a bias for the AAA lysine codon at P2, suggesting that a non-coding function for the AAA codon may be responsible for the strong bias for lysine at P2 of secretory signal sequences. We conclude that the selection for high translation initiation efficiency maybe the selective pressure that has led to codon and consequent amino acid usage at P2 of secretory proteins

Biased codon usage in signal peptides: A role in protein export

The signal peptide of proteins exported via the general secretory pathway encodes structural features that enable the targeting and export of the protein to the periplasm. Recent studies have shown biased codon usage at the second amino acid position and a high usage of non-optimal codons within the signal peptide. Altering these biases in codon usage can have deleterious effects on protein folding and export. We propose that these codon-usage biases act in concert to optimize the export process through modulating ribosome spacing on the transcript. This highlights a new aspect of protein export and implies that codon usage in the signal peptide encodes signals that are important for protein targeting and export to the periplasm

Evolution for improved secretion and fitness may be the selective pressures leading to the emergence of two NDM alleles

The New Delhi metallo-β-lactamase (NDM-1) mediates resistance to β-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-β -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new bla alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some β-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness

Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein

Non-optimal codons are generally characterised by a low concentration of isoaccepting tRNA and a slower translation rate compared to optimal codons. In a previous study, we reported a 20-fold reduction in maltose binding protein (MBP) level when the non-optimal codons in the signal sequence were optimised. In this study, we report that the 20-fold reduction is rescued when MBP is expressed at 28°C instead of 37°C, suggesting that the signal sequence optimised MBP protein (MBP-opt) may be misfolded, and is being degraded at 37°C. Consistent with this idea, transient induction of the heat shock proteases prior to MBP expression at 28°C restores the 20-fold difference, demonstrating that the difference in production levels is due to post-translational degradation of MBP-opt by the heat-shock proteases. Analysis of the structure of purified MBP-wt and MBP-opt grown at 28°C showed that although they have similar secondary structure content, MBP-opt is more resistant to thermal unfolding than is MBP-wt. The two proteins also exhibit different tryptic fragment profiles, further confirming that they are folded into conformationally different states. This is the first study to demonstrate that signal sequence non-optimal codons can influence the folding of the mature exported protein. © 2010 Elsevier B.V