At least let philosophers do the right thing when we analyze functional characterization: let no philosopher join what science has put asunder.


Table of Contents


Microorganisms are able to thrive in environments differing by their temperature, osmolarity, pH, nutrient availability, etc. by rapidly adapting their structure and physiology. The corresponding processes are based on the existence of multiple regulatory networks that control gene expression in a coordinate manner in response to environmental stimuli, and in particular to changes in the environment.

Among the many genes that coordinate gene expression in gamma-proteobacteria the hns gene is particularly important. It has been characterized as a model system for studying the regulation of bacterial physiology. In Escherichia coli, the H-NS protein is involved in numerous cellular functions, affecting the expression of genes regulated by environmental factors or required in virulence.

Our experiments showed that proteins of the H-NS family are widespread in Gram-negative bacteria. Their structural and functional organization seems to be evolutionarily conserved. In particular, they are organized in two modules: the N-terminal part involved in oligomerization is specific for a bacterial family and linked to the C-terminal DNA-binding domain by a loop. At least in E. coli and related bacteria, H-NS-like proteins play a major role in bacterial physiology by controlling the expression of numerous genes such as those involved in bacterial motility. Most of them are regulated in response to environmental stresses, which suggests a role for H-NS in the maintenance of homeostasis, an essential condition for the survival inside their host.


blueP Lejeune, P Bertin, C Walon, K Willemot, C Colson, A Danchin
A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome
Mol Gen Genet (1989) 218: 361-363 

blue P Bertin, P Lejeune, C Laurent-Winter, A Danchin
Mutations in bglY, the structural gene for the DNA-binding protein H1, affect expression of several Escherichia coli genes
Biochimie (1990) 72: 889-891 

blue P Lejeune, A Danchin
Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12
Proc Natl Acad Sci U S A (1990) 87: 360-363 

blue P Bertin, P Lejeune, C Colson, A Danchin
Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71
Mol Gen Genet (1992) 233: 184-192 

blue P Bertin, E Terao, EH Lee, P Lejeune, C Colson, A Danchin, E Collatz
The H-NS protein is involved in the biogenesis of flagella in Escherichia coli
J Bacteriol (1994) 176: 5537-5540 

blue JR Landgraf, M Levinthal, A Danchin
The role of H-NS in one carbon metabolism
Biochimie (1994) 76: 1063-1070 

blue M Levinthal, P Lejeune, A Danchin
The H-NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp, the leucine regulatory protein
Mol Gen Genet (1994) 242: 736-743 

blue A Danchin, E Krin
Filling the gap between hns and adhE in Escherichia coli K12
Microbiology (1995) 141 ( Pt 4): 959-960 

blue C Laurent-Winter, P Lejeune, A Danchin
The Escherichia coli DNA-binding protein H-NS is one of the first proteins to be synthesized after a nutritional upshift
Res Microbiol (1995) 146: 5-16 

blue C Laurent-Winter, S Ngo, A Danchin, P Bertin
Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis
Eur J Biochem (1997) 244: 767-773 

blue P Bertin, N Benhabiles, E Krin, C Laurent-Winter, C Tendeng, E Turlin, A Thomas, A Danchin, R Brasseur
The structural and functional organization of H-NS-like proteins is evolutionarily conserved in gram-negative bacteria
Mol Microbiol (1999) 31: 319-329 

blue O Soutourina, A Kolb, E Krin, C Laurent-Winter, S Rimsky, A Danchin, P Bertin
Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon
J Bacteriol (1999) 181: 7500-7508  j_bact

blue C Tendeng, C Badaut, E Krin, P Gounon, S Ngo, A Danchin, S Rimsky, P Bertin
Isolation and characterization of vicH, encoding a new pleiotropic regulator in Vibrio cholerae
J Bacteriol (2000) 182: 2026-2032  j_bact

blue P Bertin, F Hommais, E Krin, O Soutourina, C Tendeng, S Derzelle, A Danchin
H-NS and H-NS-like proteins in Gram-negative bacteria and their multiple role in the regulation of bacterial metabolism
Biochimie (2001) 83: 235-241

blue F Hommais, E Krin, C Laurent-Winter, O Soutourina, A Malpertuy, JP Le Caer, A Danchin, P Bertin
Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS
Mol Microbiol (2001) 40: 20-36  

blueF Hommais, C Laurent-Winter, V Labas, E Krin, C Tendeng, O Soutourina, A Danchin, P Bertin
Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae
Proteomics (2002) 2: 571-579 

blueOA Soutourina, E Krin, C Laurent-Winter, F Hommais, A Danchin, PN Bertin
Regulation of bacterial motility in response to low pH in Escherichia coli: the role of H-NS protein
Microbiology (2002) 148: 1543-1551  

blueC Tendeng, OA Soutourina, A Danchin, PN Bertin
MvaT proteins in Pseudomonas spp.: a novel class of H-NS-like proteins
Microbiology (2003) 149: 3047-3050  

blueC Tendeng, E Krin, OA Soutourina, A Marin, A Danchin, PN Bertin
A Novel H-NS-like protein from an antarctic psychrophilic bacterium reveals a crucial role for the N-terminal domain in thermal stability
J Biol Chem (2003) 278: 18754-18760  JBC

blueF Hommais, E Krin, JY Coppée, C Lacroix, E Yeramian, A Danchin, P Bertin
GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli
Microbiology (2004) 150: 61-72  

blueC Médigue, E Krin, G Pascal, V Barbe, A Bernsel, PN Bertin, F Cheung, S Cruveiller, S D'Amico, A Duilio, G Fang, G Feller, C Ho, S Mangenot, G Marino, J Nilsson, E Parrilli, EPC Rocha, Z Rouy, A Sekowska, ML Tutino, D Vallenet, G von Heijne, A Danchin
Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125
Genome Res (2005) 15: 1325-1335 GR biosapiens b7

blueE Krin, S Derzelle, K Bedard, M Adib-Conquy, E Turlin, P Lenormand, M-F Hullo, I Bonne, N Chakroun, C Lacroix, A Danchin
Regulatory role of UvrY in adaptation of Photorhabdus luminescens growth inside the insect
Environmental Microbiology (2008) 10: 1118–1134 

blueY Jin, RM Watt, A Danchin, JD Huang
Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli
BMC Genomics (2009) 10: 165  BMC

Control of flagellum biosynthesis

The swarming properties of E. coli hns strains are altered on semi-solid medium (Figure 1 because of a lack of flagella (Figure 2. Their synthesis requires the expression numerous genes organized in an ordered cascade. Their expression is strongly reduced in a hns mutant. This suggests that H-NS is required, directly or indirectly, in the expression of flagellum biosynthesis genes (4). Furthermore this is the first example showing that this regulatory protein may have directly or indirectly a positive control on gene expression.


Figure 1 : The presence of flagella visualized by scanning electron microscopy of wild-type (A) and hns (B) strains. (4)


Figure 2 : Motility test performed on E. coli wild-type and hns strains on semi-solid-medium. (4)

The H-NS protein affects the expression of flhDC master operon which governs all flagellar genes. This mechanism is disctinct from the activation by the CAP/cAMP complex (8), ), requires the presence of an extended untranslated 5’ region (Figure 3 and is independent of StpA protein. (10).

Figure 3: Regulatory region of flhDC master operon (8).

The nucleotides are numbered relative to the transcriptional start site (+1). The CAP consensus sequence is indicated in red. Regions protected by H-NS are underlined by blue lines. The positions of the -10 and -35 boxes are indicated in yellow. The ATG translational initiation codon and the putative ribosome binding site (RBS) are indicated in green.
In V. cholerae, overexpression of the orthologous VicH protein strongly reduces motility. This, again, suggests a role for this H-NS-like protein in the control of motility. (9)

H-NS-mediated global regulation of gene expression

In prokaryotes, the role of nucleoid-associated proteins in bacterial physiology remains largely unknown. H-NS has been initially isolated as a RNA polymerase associated factor. It is a polyanion binding protein that may have several regulatory targets

Protein profiles (Proteome analysis) and RNA expression profiles (Transcriptome analysis) of wild-type and hns strains were compared in Escherichia coli (11)

The expression of approximately 5% of the genes (Table 1) and/or the accumulation of the corresponding proteins are directly or indirectly altered in the mutant strain. About 1/5 of these genes encode proteins involved in transcription and translation, in particular many regulators. This suggests that H-NS is located at a high level in the hierarchy of regulation.(Figure 4)

One third of these genes are known or were predicted in silico to encode cell envelope components or proteins usually involved in bacterial adaptation to changes in environmental conditions, such as low pH or high osmolarity. This suggets a strong relationship between the H-NS regulon and the maintenance of intracellular homeostasis

Figure 4: Functional classification of H-NS-regulated genes.

Protein profile of a hns defective mutant in Escherichia coli

By two-dimensional electrophoresis, the synthesis and/or the accumulation of numerous proteins was shown to be specifically affected by the hns mutation (2). Many proteins were identified by microsequencing procedure (5) or, more recently, by mass spectrometry (11). Most of them are involved in the adaptation of microorganisms to environmental stresses such as temperature, pH or osmolarity.

Figure 5 : 2D gel electrophoresis of proteins extracted from wild-type (A) and hns (B) E. coli strains . (11)

In comparison with its wild-type counterpart, the accumulation level of proteins in green is reduced in hns strain while proteins in red are preferentially accumulated.

Structure-function-evolution of H-NS-like proteins

The three-dimensional structure of H-NS protein has not been yet determined, excepted for the C-terminal domain. Nevertheless, in silico analysis suggests that this protein contains 2 domains: the N-terminal part is predicted to be mainly a-helical and to adopt a coiled-coil structure which could play a role in oligomerisation and the C-terminal DNA-binding domain which is predicted as a mixed a-b structure. (Figure 6)
Figure 6: Prediction of secondary structure by the MLRC method (A) and of coiled-coil structure by the COILS program (B).
In silico analysis performed on H-NS-like proteins of various microorganisms (6,7,9,10) but also in vivo experiments (complementation of phenotypic alterations associated with a hns mutation in E. coli) suggest that they are structurally related (7).
The C-terminal part which is the most conserved region has been demonstrated to interact with DNA (Figure 7).

All proteins show a preferential binding to curved DNA and, despite a low conservation in their N-terminal part, an ability to dimerize in vitro. These results demonstrate that a family of proteins structurally and functionally related to H-NS of E. coli exists in microorganisms, at least in Gram-negative bacteria (6,7,9,10).
Figure 7: Amino acid sequence alignment of the C-terminal domain of H-NS and related proteins. Strictly conserved residues are indicated in red. (10)