A putative curved DNA region upstream of rcsA in Escherichia coli plays a key role in transcriptional regulation by H‐NS

It is well established that in Escherichia coli, the histone‐like nucleoid structuring (H‐NS) protein also functions as negative regulator of rcsA transcription. However, the exact mode of regulation of rcsA transcription by H‐NS has not been studied extensively. Here, we report the multicopy effect of dominant‐negative hns alleles on the transcription of rcsA based on expression of cps‐lac transcriptional fusion in ∆lon, ∆lon rpoB12, ∆lon rpoB77 and lon + strains. Our results indicate that H‐NS defective in recognizing curved DNA fails to repress rcsA transcription significantly, while nonoligomeric H‐NS molecules still retain the repressor activity to an appreciable extent. Together with bioinformatics analysis, our study envisages a critical role for the putative curved DNA region present upstream of rcsA promoter in the transcriptional regulation of rcsA by H‐NS.

It is well established that in Escherichia coli, the histone-like nucleoid structuring (H-NS) protein also functions as negative regulator of rcsA transcription. However, the exact mode of regulation of rcsA transcription by H-NS has not been studied extensively. Here, we report the multicopy effect of dominant-negative hns alleles on the transcription of rcsA based on expression of cps-lac transcriptional fusion in Δlon, Δlon rpoB12, Δlon rpoB77 and lon + strains. Our results indicate that H-NS defective in recognizing curved DNA fails to repress rcsA transcription significantly, while nonoligomeric H-NS molecules still retain the repressor activity to an appreciable extent. Together with bioinformatics analysis, our study envisages a critical role for the putative curved DNA region present upstream of rcsA promoter in the transcriptional regulation of rcsA by H-NS.
In Escherichia coli, the genetic material is organized in the form of nucleoid and the DNA-binding proteins such as histone-like proteins serve as a dynamic scaffold for nucleoid organization [1][2][3][4]. The histone-like nucleoid structuring (H-NS, previously denoted as H1) protein of E. coli is one of the major components of the nucleoid. hns gene was identified by Pon et al. [5], and it maps at 27 min of E. coli chromosome. H-NS protein comprises 137 amino acids with 15.5 kDa molecular weight. Although initial studies suggested that H-NS is involved only in the organization of chromosome, the identification that H-NS has higher propensity to bind to DNA, especially the AT-rich sequences, clearly indicated the regulatory function associated with H-NS [6][7][8]. H-NS was found to affect gene expression in a number of different ways, and it has been reported that expression of over 5% of the E. coli genes is affected in hns mutant [9][10][11].
H-NS binding does not seem to occur with any obvious sequence specificity [12,13]. Different mechanisms for transcriptional regulation by H-NS have been proposed; the most accepted models are as follows: H-NS might indirectly regulate initiation by binding to region distal from the promoter which causes change in supercoiling that in turn affects the supercoiling-sensitive promoters; and H-NS can also directly inhibit transcription by preferential binding to the promoter region. Many of the preferred H-NS binding sites contain an A/T-rich region, suggesting that a sequence-induced curvature is causing the preferential binding [14][15][16][17][18]. Studies on structural aspects of H-NS revealed that H-NS is comprised of a C-terminal DNA-binding domain and a coiled-coil N-terminal domain that mediates oligomerization, forming higher-order homomeric or heteromeric complexes. At least two dimerization sites have been identified that allow H-NS to form higher-order oligomers [19][20][21][22]. The oligomerization and DNA-binding domains are  joined via a flexible linker. H-NS itself acts as a repressor for its own promoter, and apart from H-NS, StpA, Fis and CspA also play a role in the regulation of H-NS expression [23,24]. There is also a post-transcriptional negative regulatory mechanism which involves a small RNA called DsrA and an RNA-binding chaperon protein called Hfq [25,26]. The expression of H-NS is also increased by an unknown mechanism during growth at elevated hydrostatic pressure. H-NS and other nucleoid-associated proteins can recognize horizontally acquired DNA and transcriptionally silence it through xenogeneic silencing under environmental conditions that do not require expression of horizontally acquired genes [27,28]. In addition to its role in nucleoid architecture, H-NS plays a pleiotropic role in bacterial response to environmental stimuli such as starvation and changes in pH, temperature and osmolarity [29][30][31].
Very recently, we have reported the suppression of overexpression of genes implicated in colanic acid capsular polysaccharide (Cps) synthesis in Δlon mutant of E. coli by two novel rpoB mutations, namely rpoB12 and rpoB77. Genetic and molecular analyses clearly showed that downregulation of rcsA transcription is the primary reason for the elicitation of this capsule expression suppression (Ces) phenotype by these two rif alleles. Furthermore, our study clearly indicated that the presence of functional H-NS is mandatory for both the rpoB mutations to function as capsule expression suppressors in the Δlon strain of E. coli [32]. Sledjesky and Gottesman [25] have shown that H-NS functions as a repressor for rcsA transcription, and their study also revealed the involvement of a small RNA, namely DsrA located downstream of rcsA, in the regulation of rcsA by H-NS. DsrA binds to H-NS and thereby inhibits the action of H-NS on rcsA transcription. However, the mode of binding and the exact binding region for H-NS in the promoter region of rcsA have not been reported so far. They have suggested that the upstream region of rcsA promoter might possess bending/curved DNA region [25]. In our earlier study, we have provided evidence for the occurrence of bendable DNA region upstream of rcsA promoter through bioinformatics analyses [32]. In this study, perhaps for the first time we have given the genetic evidence that supports the presence of putative curved DNA region upstream of rcsA promoter. Furthermore, we have shown that the H-NS molecule which is defective in the formation of higher-order oligomers can still function as a repressor at the rcsA promoter. The bioinformatics analyses show that the region around 400 bp upstream of rcsA promoter might serve as H-NS binding site.

Materials and methods
Media composition, chemicals, fine chemicals and genetic and molecular techniques used in this study The media (conventional LB and minimal media) composition used in this entire study is essentially as described in Ref. [33]. Materials used for media, buffer, solutions, most of the antibiotics and other fine chemicals were purchased from HiMedia, India. Streptomycin was purchased from Sarabhai Chemicals, India, and the final concentration of each of them is quoted wherever appropriate. All the genetic techniques were according to Ref. [33] (with minor modifications), and molecular techniques employed in this study were as per Ref. [34]. Table 1 gives the list of bacterial strains, phages and plasmids used in this study. All the bacterial strains are the derivatives of E. coli K-12, and the genetic nomenclature is according to Refs [35] and [36].

Plasmid isolation, transformation and construction of strains bearing clones of dominant-negative alleles of hns
The strains bearing the plasmids harbouring the dominantnegative alleles of hns, namely pLGhns-Δ64, pLGhns-P116S, pLGhns-T55P, pLGhns-L26P and pLGhns + , were procured from J Gowrishankar, CDFD, Hyderabad, India. The plasmids were isolated from relevant strains using the alkaline lysis method [37], and the presence of insert was confirmed through restriction digestion analyses. The strains, namely SG20780 (Δlon cps-lac), SG201781 (lon + cps-lac), MMRT6 (Δlon cps-lac rpoB12) and MMRT23 (Δlon cps-lac rpoB77), were transformed with the relevant clones. The CaCl 2 -mediated transformation technique was followed. As the vector backbone (pLG339) bears kanamycin as selection marker, the transformants were selected on LB plates containing kanamycin. Representative transformants from each case were selected and purified for further use.
Beta-galactosidase assay 0.1 mL of overnight cultures of each strain (carrying cps-lac fusion) was subcultured into 5 mL of M9 minimal medium containing glucose as carbon source and grown at 30°C. The cultures were allowed to attain mid-log phase, and then, the optical density of the cultures was recorded at 600 nm wavelength. The beta-galactosidase expressed from cps-lac fusion was assayed as described in Ref. [33] with minor modifications.

Bioinformatics analyses
The DNA sequence of the coding region of rcsA including the upstream region of rcsA promoter (till -600) was retrieved from Ecocyc.org, and the structure of the DNA sequence was elucidated using the software MODEL.IT (http://hydra.icgeb.trieste.it/dna/index.php). Further analyses/manipulations of the structure were carried out using PYMOL (http://pymol.org/edu/?q=educational).

H-NS which is defective in recognizing curved DNA fails to repress rcsA transcription
In our earlier study pertaining to the isolation and characterization of novel rpoB mutations capable of suppressing the overproduction of colanic acid Cps in lon mutant of E. coli, we have substantiated the role of functional H-NS in the elicitation of Ces phenotype by the two rpoB mutations, namely rpoB12 (C 1576 to T 1576 ; His526 to Tyr526) and rpoB77 (C 1535 to T 1535 ; Ser512 to Tyr512) [32]. As a continuation to this aspect, the effect of dominant-negative alleles of hns in Ces strains (Δlon rpoB12 and Δlon rpoB77) and in parental strains (Δlon and lon + ) was studied (strains bearing the dominant-negative hns alleles were procured from Gowrishankar, CDFD, India). For information of different dominant-negative alleles of hns used in this study ( Table 2). All the hns alleles, namely hnsP116S, hnsΔ64, hnsT55P and hnsL26P, have been cloned into a vector with its native promoter. The clone bearing hns + (pLG-hns + ) was also used, as in this case the result could be presumed and it can be used for better comparison. All the clones were 323 As was expected in the absence of any clone, in the Δlon strain a higher level of expression of cps-lac was seen. However, in Δlon rpoB12 and Δlon rpoB77 mutants due to elicitation of Ces phenotype, the cps-lac expression was less as was expected. In the lon + strain, in accordance with expectation, a very low level of expression of cps-lac was seen due to RcsA degradation transformed into the relevant strains, namely SG20780 (Δlon cps-lac), SG20781 (lon + cps-lac), MMRT6 (Δlon cps-lac rpoB12) and MMRT23 (Δlon cps-lac rpoB77).
In each case, a transformant was purified, cells were grown overnight in minimal glucose medium containing kanamycin till mid-log phase and b-galactosidase assay was carried out as described in Materials and methods. As was expected, introduction of the hns + clone reduced the expression of cps-lac fusion to an appreciable degree in all the strains (Fig. 1). These results once again signify the role of H-NS as a repressor of rcsA transcription. As shown in Fig. 1, introduction of clones bearing variant alleles of hns, namely pLGhns-P116S and pLGhns-Δ64, into the relevant strains revealed the following: with reference to the mutant H-NS (with P116S amino acid substitution) which is defective in binding to curved DNA, the expression level of cps-lac has gone up to appreciable levels in all the above-mentioned strains. Comparative analyses of the multicopy effect of hns + and hnsP116S alleles on the expression of cps-lac fusion in the relevant strains clearly show the inability of the mutant form of H-NSP116S molecules to exert complete repressor activity like the wild-type although it is reported to retain nonspecific DNA-binding activity (for the comparative values, see Table 2). These results strongly support the view that the upstream region of rcsA promoter is likely to contain a bendable/curved region and the inability of mutant form of H-NS (H-NSP116S) to bind to such putative bendable/curved region of rcsA promoter could be the cause for higher level of cps-lac expression in the relevant strains. Introduction of pLG-hnsΔ64 increased the cps-lac expression to some extent that clearly implies that the H-NS molecules bearing only the N-terminal region are not completely defective in repression at rcsA promoter.
The hns alleles cloned into these vectors result in the amino acid substitution at N-terminal region (at amino acid positions T55P and L26P), which is expected to affect the oligomerization property of H-NS molecules. This indirectly but strongly supports the view that even the oligomerization-defective H-NS could interfere with cps-lac expression perhaps by repressing rcsA transcription (Fig. 1).
Bioinformatics analyses reveal that H-NS binding region could be present~400 bp upstream of rcsA promoter H-NS has been shown to bind to the curved DNA region preferentially [14][15][16]. In our earlier report, we have given evidence for the involvement of functional H-NS in the elicitation of Ces phenotype by rpoB12 and rpoB77 mutations, and we have predicted the presence of bendable DNA sequence upstream of the rcsA promoter [32]. Here, we show that the DNA sequence around 400 bp upstream of the rcsA promoter is probably bendable in nature. Using the software MODEL.IT, we have modelled the DNA region present upstream of rcsA, and the image was further manipulated by PYMOL software. Figure 2A,B clearly shows that the region -130 to -400 does exist as a putative curved DNA. Therefore, the sequence underlined in Fig. 2C could be the most probable region where the H-NS might bind to and repress rcsA transcription.

Discussion
Overproduction of colanic acid Cps and extreme sensitivity to DNA-damaging agents are considered as the iconic phenotypes of a lon mutant of E. coli. Detailed study on these aspects revealed that stabilization of two Lon substrates, namely RcsA (the positive regulator of cps transcription) and SulA (cell division inhibitor that gets induced upon DNA damage), is the main reason for the elicitation of the above-mentioned phenotypes, respectively [38][39][40]. Previous studies pertaining to isolation of suppressor(s) for these two hallmark phenotypes implicated a vital role of mutation in ssrA and a novel allele of dnaJ (faa) [41][42][43][44]. Earlier using an unorthodox, wee bit strategy, we sought for rif (rpoB) mutations capable of suppressing either one or both of the phenotypes of lon mutant.
In such an attempt, we were indeed successful in isolating two such novel rif alleles (rpoB12 and rpoB77) that could suppress only the overproduction of capsule synthesis. Detailed analyses showed that the elicitation of this Ces phenotype by these rif mutations primarily stems from the downregulation of rcsA transcription. Our study also revealed the requirement of functional H-NS in the elicitation of Ces phenotype [32]. oligomerization [8]. During the course of such analyses, dominant-negative variants of hns have been isolated [19,20]. In this study, the effect of different clones bearing dominant-negative alleles of hns such as hnsP116S, hnsT55P, hnsL26P, hnsΔ64 and hns + , in phenotypically Ces strains such as MMRT6 (Δlon rpoB12) and MMRT23 (Δlon rpoB77) and also in Δlon and lon + strains has clearly revealed that the hnsP116S allele that codes for H-NS but is defective in recognizing curved DNA almost completely abolished the elicitation of Ces phenotype in both Δlon rpoB12 and Δlon rpoB77 strains. This effect was seen even in Δlon and lon + strains. This indirectly implies that the region upstream of rcsA promoter might possess putative curvature which might play an important role in the regulation of rcsA transcription by H-NS.
Further, the clones bearing hns alleles coding for the amino acid substitutions, namely T55P and L26P (which are defective in the formation of higher-order oligomers), significantly reduced the cps-lac expression not only in the Δlon rpoB12 and Δlon rpoB77 strains but also in the Δlon and lon + strains; that is, the effect is albeit closer to that of wild-type H-NS. This was totally unexpected as we imagined that the mutant forms of H-NS cannot form higher-order oligomers and therefore will not be able to repress rcsA transcription. But the fact that we have made such an observation compelled us to make a model that in a nonoligomeric state and even without forming higherorder oligomers, these mutant forms of H-NS perhaps might be able to bind to DNA and function as repressors for rcsA transcription. Williams et al. [19] have reported that the introduction of clone(s) bearing oligomerization-defective hns alleles, namely L26P and T55P, drastically decreased the expression of semisynthetic 5A6Agal promoter. Similar analyses with one other H-NS-regulated gene, namely proU, indicate that the expression of its promoter was not found to be identical to that of 5A6Agal promoter. These observations signify the fact that the regulatory function of H-NS depends on the sequence features of the promoters also. In similar analyses, it was also found that introduction of clone bearing hns allele (P116S) elevated the expression of 5A6Agal promoter to an appreciable degree, while the same was once again not found to be true with proU promoter. It has been reported that the upstream region of 5A6Agal promoter bears a curvature [45]. However, in the case of proU, the presence of any curved DNA region has not been reported and notably the repression by H-NS essentially needs extensive nucleoprotein formation at the proU promoter [45,46], which perhaps gives a clue about H-NS binding-induced DNA bending at the proU promoter region.
The expression pattern of 5A6Agal promoter and rcsA promoter is found to be similar in the presence of different dominant-negative hns alleles. These observations clearly favour the notion that the upstream region of rcsA might possess curved DNA which would serve as binding region for H-NS, thus aiding H-NS to transcriptionally regulate rcsA expression.