Christine Laurent-Winter

Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS

Despite many years of intense work investigating the function of nucleoid‐associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown. The two‐dimensional protein patterns were compared and expression profiling was carried out on H‐NS‐deficient and wild‐type strains of Escherichia coli K‐12. The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain. About one‐fifth of these genes encode proteins that are involved in transcription or translation and one‐third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions. The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild‐type strain. In particular, the putative regulator, YhiX, plays a central role in the H‐NS control of genes required in the glutamate‐dependent acid stress response. These results suggest that there is a strong relationship between the H‐NS regulon and the maintenance of intracellular homeostasis.

Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope.

The antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non‐covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall‐linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.

The structural and functional organization of H‐NS‐like proteins is evolutionarily conserved in Gram‐negative bacteria

The structural gene of the H‐NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae. Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H‐NS, particularly in the N‐terminal domain. To obtain information on the structure, function and/or evolution of H‐NS, we searched for other H‐NS‐related proteins in the latest databases. We found that HvrA, a trans‐activator protein in Rhodobacter capsulatus, has a low but significant similarity with H‐NS and H‐NS‐like proteins. This Gram‐negative bacterium is phylogenetically distant from Escherichia coli. Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H‐NS, StpA (an H‐NS‐like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H‐NS‐related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides, XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae. These results demonstrate that proteins structurally and functionally related to H‐NS are widespread in Gram‐negative bacteria.

Regulation of IκBβ Degradation SIMILARITIES TO AND DIFFERENCES FROM IκBα

The transcription factor NF-κB (nuclear factor-κB) is neutralized in nonstimulated cells through cytoplasmic retention by IκB inhibitors. In mammalian cells, two major forms of IκB proteins, IκBα and IκBβ, have been identified. Upon treatment with a large variety of inducers, IκBα and IκBβ are proteolytically degraded, resulting in NF-κB translocation into the nucleus. Recent observations suggest that phosphorylation of serines 32 and 36 and subsequent ubiquitination of lysines 21 and 22 of IκBα control its signal-induced degradation. In this study we provide evidence that critical residues in the NH2-terminal region of IκBβ (serines 19 and 23) as well as its COOH-terminal PEST region control IκBβ proteolysis. However Lys-9, the unique lysine residue in the NH2-terminal region of IκBβ, is not absolutely required for its degradation. We also demonstrate that following stimulation, an underphosphorylated nondegradable form of IκBβ accumulates. Surprisingly, our data suggest that unlike IκBα, IκBβ is constitutively phosphorylated on one or two of the critical NH2-terminal serine residues. Thus, phosphorylation of these sites is necessary for degradation but does not necessarily constitute the signal-induced event that targets the molecule for proteolysis.

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus

Two‐dimensional gel electrophoresis was used to follow low changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse‐labelled with [35S]‐methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two‐dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brp4‐dependent proteins were mapped to a 10 kb Sstl fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton‐inducible tipA promoter. N‐terminal amino acid sequences of two brpA‐dependent proteins, obtained by direct microsequencing of protein spots excised from two‐dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA‐dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5‐29, including 16 brpA‐dependent ones, identified 75 families having distinct patterns of expression. Many brpA‐dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA‐independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.

Mutations in Bgly, the Structural Gene for the Dna-binding Protein H1, Affect Expression of Several Escherichia-coli Genes

The protein patterns of a bglY mutant and the isogenic wild-type strains were compared on 2-dimensional gel electrophoresis. The synthesis of at least 36 peptides was affected. This suggests a global but specific role on gene expression for the DNA-binding protein H1.

Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function

Structural genomics is a new approach in functional assignment of proteins identified via whole‐genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two‐dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide‐binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP‐binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP‐binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand‐binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a β‐sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the β‐sheets is mostly involved in biological activity.

Transcription Regulation Coupling of the Divergent argG and metY Promoters in Escherichia coli K-12

The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number of Escherichia coli genes, including those involved in carbon metabolism. We identified two new targets of this complex: argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, and metY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activates argG transcription and inhibits metY transcription from the same DNA position. We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation of metY mediated by CAP. The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.

Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae.

In this paper, we initiated the first two‐dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.

The Escherichia coli DNA-binding protein H-NS is one of the first proteins to be synthesized after a nutritional upshift

Using two-dimensional electrophoresis, the patterns of polypeptide expression of a wild type and an hns mutant of Escherichia coli were examined in exponential and late stationary phases. The same procedure was used for a kinetic study of proteins synthesized during the first 60 min following inoculation into fresh complete medium. The present study focusses on 35 polypeptides differentially synthesized according to the strain and/or the growth phase. One of the most striking and unexpected observations in this work was a strong and transient synthesis of H-NS in the wild-type strain just after the nutritional upshift.