Philippe Bertin

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.

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. coliu200a), 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 bacterial motility in response to low pH in Escherichia coli: the role of H-NS protein.

The effect of detrimental conditions on bacterial motility in Escherichia coli was investigated. Expression profiling of mutant E. coli strains by DNA arrays and analysis of phenotypic traits demonstrated that motility and low-pH resistance are coordinately regulated. Analysis of transcriptional fusions suggests that bacterial motility in response to an acidic environment is mediated via the control by H-NS of flhDC expression. Moreover, the results suggested that the presence of an extended mRNA 5 end and DNA topology are required in this process. Finally, the presence of a similar regulatory region in several Gram-negative bacteria implies that this mechanism is largely conserved.

Characterization of polyamine synthesis pathway in Bacillus subtilis 168

The ubiquitous polyamines fulfil a variety of functions in all three kingdoms of life. However, little is known about the biosynthesis of these compounds in Gram‐positive bacteria. We show that, in Bacillus subtilisthere is a single pathway to polyamines, starting from arginine, with agmatine as an intermediate. We first identified the structural gene of arginine decarboxylase, speA (formerly cadu200a), and then described the speE speB operon, directing synthesis of spermidine synthase and agmatinase. This operon is transcribed into two messenger RNAs, a major one for the speE gene and a minor one for both speE and speB. The promoter of the operon was identified upstream from the speE gene by primer extension analysis. Transcription of this operon indicated that the level of agmatinase synthesis is very low, thus allowing a stringent control on the synthesis of putrescine and, therefore, of all polyamines. This is consistent with the level of polyamines measured in the cell.

H-NS and H-NS-like proteins in Gram-negative bacteria andtheir multiple role in the regulation of bacterial metabolism

In Escherichia coli, the H-NS protein plays an important role in the structure and the functioning of bacterial chromosome. A homologous protein has also been identified in several enteric bacteria and in closely related organisms such as Haemophilus influenzae. To get information on their structure and their function, we identified H-NS-like proteins in various microorganisms by different procedures. In silico analysis of their amino acid sequence and/or in vivo experiments provide evidence that more than 20 proteins belong to the same class of regulatory proteins. Moreover, large scale technologies demonstrate that, at least in E. coli, the loss of motility in hns mutants results from a lack of flagellin biosynthesis, due to the in vivo repression of flagellar gene expression. In contrast, several genes involved in adaptation to low pH are strongly induced in a H-NS deficient strain, resulting in an increased resistance to acidic stress. Finally, expression profiling and phenotypic analysis suggest that, unlike H-NS, its paralogous protein StpA does not play any role in these processes.

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.

Control of bacterial motility by environmental factors in polarly flagellated and peritrichous bacteria isolated from Lake Baikal

ABSTRACT Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA genes showed that these strains belong to two distinct genera, i.e., Enterobacter andPseudomonas; they were named strains 22 and Y1000, respectively. Both strains swarmed at 25°C and remained motile at low temperatures (4°C), especially the Pseudomonasstrain, which further supports the psychrotrophic characteristics of this strain. In contrast, a strong inhibition of motility was observed at above 30°C and with a high NaCl concentration. The existence of flagellar regulatory proteins FlhDC and FleQ was demonstrated inEnterobacter strain 22 and Pseudomonasstrain Y1000, respectively, and environmental conditions reduced the expression of the structural genes potentially located at the first level in the flagellar cascade in both organisms. Finally, as inEnterobacter strain 22, a strong reduction in the transcription of the master regulatory gene fleQ was observed in Pseudomonas strain Y1000 in the presence of novobiocin, a DNA gyrase inhibitor, suggesting a link between DNA supercoiling and motility control by environmental factors. Thus, striking similarities observed in the two organisms suggest that these processes have evolved toward a similar regulatory mechanism in polarly flagellated and laterally flagellated (peritrichous) bacteria.

Characterization of BpH3, an H-NS-like protein in Bordetella pertussis

This study describes the characterization of BpH3, a Bordetella pertussis DNA‐binding protein. Sequence analysis reveals significant homology with the H‐NS sequence of Escherichia coli and Haemophilus influenzae, particularly in the C‐terminal part of the proteins. Our results provide evidence that H‐NS and BpH3 display functional homology. First, expression of BpH3 in an hns mutant results in restoration of motility, an H‐NS‐dependent phenotype. This effect is dependent on the level of BpH3 expression and results from transcriptional activation of the flagellar master operon. Second, the high level of β‐glucosidase associated with hns mutations is reversed to the low wild‐type level in the presence of BpH3. Third, BpH3 is able, like H‐NS, to preferentially bind in vitro to curved DNA fragments, such as flhDC and bla promoter regions. Our results are the first demonstration that proteins homologous to H‐NS exist in bacteria phylogenetically distant from H. influenzae and enterobacteria.

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 regulation of Enzyme IIA Glc expression controls adenylate cyclase activity in Escherichia coli

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.