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Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.

The crystal structure of cold-shock protein E from S. typhimurium (StCspE) has been determined at 1.1 Å resolution.

In prokaryotic organisms, cold shock triggers the production of a small highly conserved family of cold-shock proteins (CSPs). CSPs have been well studied structurally and functionally in Escherichia coli and Bacillus subtilis, but Salmonella typhimurium CSPs remain relatively uncharacterized. In S. typhi­­murium, six homologous CSPs have been identified: StCspA–E and StCspH. The crystal structure of cold-shock protein E from S. typhimurium (StCspE) has been determined at 1.1 Å resolution and has an R factor of 0.203 after refinement. The three-dimensional structure is similar to those of previously determined CSPs and is composed of five antiparallel β-strands forming a classic OB fold/five-stranded β-barrel. This first structure of a CSP from S. typhimurium provides new insight into the cold-shock response of this bacterium.

This paper describes the application of our distributed computing framework for crystal structure prediction (CSP), Modified Genetic Algorithms for Crystal and Cluster Prediction (MGAC) to predict the crystal structure of flexible molecules using the General Amber Force Field (GAFF) and the CHARMM program. The MGAC distributed computing framework which includes a series of tightly integrated computer programs for generating the molecule’s force field, sampling crystal structures using a distributed parallel genetic algorithm, local energy minimization of the structures followed by the classifying, sorting and archiving of the most relevant structures. Our results indicate that the method can consistently find the experimentally known crystal structures of flexible molecules, but the number of missing structures and poor ranking observed in some crystals show the need for further improvement of the potential.

The title compound, C14H9N, is the second crystallographically characterized example of an ynamine with an H atom in the C-terminal position. There are two independent mol­ecules (A and B) in the asymmetric unit. The structures of both mol­ecules are essentially planar (r.m.s. deviation = 0.0312 and 0.0152 Å). The N—Csp bond lengths are 1.353 (4) and 1.350 (4) Å, and those of the acetyl­ene bonds are 1.189 (4) and 1.190 (4) Å. The Csp—H bond lengths are 0.95 (5) and 0.97 (4) Å. These geometries are consistent with those of the previously reported ynamine characterized by crystallography. In the crystal, the mol­ecules stack along the c axis, forming two kinds of columnar structures. The acetyl­ene C atoms of mol­ecule A have a short contact [3.341 (4) Å and 3.396 (4) Å] with an adjacent mol­ecule A at the C—C bond of the fused part, which originates in π–π stacking inter­action; no remarkable spatial contact is recognized within the stacking of mol­ecule B.

The title diphenyl­arsino compound, C30H32As2 or Ph2As(CH2)6AsPh2, lies about a crystallographic inversion centre located at the mid-point of the central Csp 3—Csp 3 bond of the methyl­ene chain. The two benzene rings bonded to As are inclined to one another at a dihedral angle of 75.98 (8)°. In the crystal structure, weak inter­molecular C—H⋯π inter­actions stack the mol­ecules down the b axis.

In the absence of three-dimensional (3D) structures of potential drug targets, ligand-based drug design is one of the popular approaches for drug discovery and lead optimization. 3D structure-activity relationships (3D QSAR) and pharmacophore modeling are the most important and widely used tools in ligand-based drug design that can provide crucial insights into the nature of the interactions between drug target and ligand molecule and provide predictive models suitable for lead compound optimization. This review article will briefly discuss the features and potential application of recent advances in ligand-based drug design, with emphasis on a detailed description of a novel 3D QSAR method based on the conformationally sample pharmacophore (CSP) approach (denoted CSP-SAR). In addition, data from a published study is used to compare the CSP-SAR approach to the Catalyst method, emphasizing the utility of the CSP approach for ligand-based model development.

Increased frequency of cavum septum pellucidum (CSP) has been inconsistently observed in schizophrenia, and little is known about its functional implications. We investigated whether patients with schizophrenia were more likely than healthy controls to have CSP, and among patients assessed the relationship between CSP, psychiatric symptoms, and selected neuropsychological functions. Seventy-seven patients with diagnoses of DSM-IV schizophrenia spectrum disorders and 55 healthy controls were studied and completed a 1.5 T MRI scan. Two raters, blind to group membership, determined the presence, length and grade of the CSP. A subset of participants also underwent neuropsychological testing. A CSP of at least 1 mm in length was present in 68.8% of patients and 76.4% of controls, and the groups did not differ significantly with respect to presence or absence, length, overall size, or percent with an abnormally large CSP (≥ 6 mm). Patients with an abnormally large CSP demonstrated poorer performance on measures of verbal learning and memory than patients with smaller CSP. Among patients, CSP length was significantly correlated with negative symptoms, verbal learning, and sentence comprehension. Among patients with abnormally large CSP, CSP length was correlated with reaction time on two conditions of a Continuous Performance Test. CSP, while prevalent, was not more frequent in our sample of patients with schizophrenia, and had few associations with symptom severity or neuropsychological deficits.

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37°C to 10°C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.

The structure of the title compound, C13H14O2, a penta­cyclo­undecane cage derivative, exhibits unusual Csp 3—Csp 3 single-bond lengths ranging from 1.505 (3) to 1.607 (2) Å and strained bond angles as small as 88.7 (1)° and as large as 121.0 (2)°. In this meso compound, an inter­nal non-crystallographic mirror plane exists, bis­ecting the mol­ecule. In the crystal, weak C—H⋯O hydrogen bonds link the mol­ecules into an infinite spiral about a twofold screw axis along the [100] direction.

The title mol­ecule, C18H24O12, has crystallographic 2/m symmetry with two acetate group located on a mirror plane. The H—Csp 3—O—Csp 2 torsion angles characterizing orientation of the acetyl groups with respect to the cyclo­hexane ring are 0.0, 23.9 and −23.9°. The cyclo­hexane ring is in a chair conformation with all substituents in equatorial positions. In the crystal, mol­ecules are connected through C—H⋯O hydrogen bonds into a chain extending along the c axis.

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.

Escherichia coli contains nine members of the CspA family. CspA and some of its homologues play critical role in cold acclimation of cells by acting as RNA chaperones, destabilizing nucleicacid secondary structures. Disruption of nucleic acid melting activity of CspE led to loss of its transcription antitermination activity and consequently its cold acclimation activity. To date, the melting activity of Csp proteins was studied using partially double-stranded model nucleic acids substrates forming stem–loop structures. Here, we studied the mechanism of nucleic acid melting by CspE. We show that CspE melts the stem region in two directions, that CspE-induced melting does not require the continuity of the substrate's loop region, and CspE can efficiently melt model substrates with single-stranded overhangs as short as 4 nt. We further show that preferential binding of CspE at the stem–loop junction site initiates melting; binding of additional CspE molecules that fully cover the single-stranded region of a melting substrate leads to complete melting of the stem.

The title compound, C72F36, is one of four isomers of C60(CF3)12 for which crystal structures have been obtained. The fullerene mol­ecule has an idealized Ih C60 core with the 12 CF3 groups arranged in an asymmetric fashion on two ribbons of edge-sharing C6(CF3)2 hexa­gons, a para–meta–para–para–para–meta–para ribbon and a para–meta–para ribbon, giving an overall pmp 3 mp,pmp structure. There are no cage Csp 3—Csp 3 bonds. The F atoms of two CF3 groups are disordered over two positions; the site occupancy factors are 0.85/0.15 and 0.73/0.27. There are intra­molecular F⋯F contacts between pairs of CF3 groups on the same hexa­gon that range from 2.521 (3) to 2.738 (4) Å.

The three-dimensional structures of two odorant binding proteins (OBPs) and one chemosensory protein (CSP) from a polyphagous ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) were resolved bioinformatically. The results show that both SguaOBP1 and OBP2 are classic OBPs, whereas SguaCSP1 belongs to non-classic CSPs which are considered as the “Plus-C” CSP in this report. The structural differences between the two OBPs and between OBP and CSP are thoroughly described, and the structural and functional significance of the divergent C-terminal regions (e.g., the prolonged C-terminal region in SguaOBP2 and the additional pair of cysteines in SguaCSP1) are discussed. The immunoblot analyses with antisera raised against recombinant SguaOBP1, OBP2, and CSP1, respectively, indicate that two SguaOBPs are specific to antennae, whereas SguaCSP1, which are more abundant than OBPs and detected in both male and female wasps, expresses ubiquitously across different tissues.

We also describe the ultrastructure of the antennal sensilla types in S. guani and compare them to 19 species of parasitic Hymenoptera. There are 11 types of sensilla in the flagellum and pedicel segments of antennae in both male and female wasps. Seven of them, including sensilla placodea (SP), long sensilla basiconica (LSB), sensilla coeloconica (SC), two types of double-walled wall pore sensilla (DWPS-I and DWPS-II), and two types of sensilla trichodea (ST-I and ST-II), are multiporous chemosensilla. The ultralsturctures of these sensilla are morphologically characterized. In comparison to monophagous specialists, the highly polyphagous generalist ectoparasitoids such as S. guani possess more diverse sensilla types which are likely related to their broad host ranges and complex life styles. Our immunocytochemistry study demonstrated that each of the seven sensilla immunoreacts with at least one antiserum against SguaOBP1, OBP2, and CSP1, respectively. Anti-OBP2 is specifically labeled in DWPS-II, whereas the anti-OBP1 shows a broad spectrum of immunoactivity toward four different sensilla (LSB, SP, ST-I and ST-II). On the other hand, anti-CSP1 is immunoactive toward SP, DWPS-I and SC. Interestingly, a cross co-localization pattern between SguaOBP1 and CSP1 is documented for the first time. Given that the numbers of OBPs and CSPs in many insect species greatly outnumber their antennal sensilla types, it is germane to suggest such phenomenon could be the rule rather than the exception.

In the title mol­ecule, C10H11NO2, the benzene ring forms dihedral angles of 33.15 (2) and 6.20 (2)° with the mean planes of the amide and propen­oxy groups, respectively. The amide –NH2 group is oriented toward the propen­oxy substituent and forms a weak intra­molecular N—H⋯O hydrogen bond to the propen­oxy O atom. The conformation of the propen­oxy group at the Csp 2—Csp 3 and Csp 3—O bonds is synperiplanar and anti­periplanar, respectively. In the crystal, N—H⋯O hydrogen bonds involving the amide groups generate C(4) and R 2 3(7) motifs that organize the mol­ecules into tapes along the a-axis direction. There are C—H⋯π inter­actions between the propen­oxy –CH2 group and the aromatic system of neighboring mol­ecules within the tape. The mean planes of the aromatic ring and the propen­oxy group belonging to mol­ecules located on opposite sites of the tape form an angle of 83.16 (2)°.

The title compound, C10H11NTe, is the first organyl ethynyl telluride, R—Te—C C—H, to be structurally characterized. In the L-shaped mol­ecule, the aryl moiety, viz. Me2NC6H4Te, is almost perpendicular to the Te—C C—H fragment. The Te—Csp 2 bond [2.115 (3) Å] is significantly longer than the Te—Csp bond [2.041 (4) Å]. The Te—C C group is approximately linear [Te—C—C = 178.5 (4)° and C C = 1.161 (5) Å], while the coordination at the Te atom is angular [C—Te—C = 95.92 (14)°]. In the crystal structure, there are Csp—H⋯N hydrogen bonds which are perpendicular to the CNMe2 group; the N atom displays some degree of pyramidalization. Centrosymmetrically related pairs of mol­ecules are linked by Te⋯π(ar­yl) inter­actions, with Te⋯Cg = 3.683 (4) Å and Csp—Te⋯Cg = 159.1 (2)° (Cg is the centroid of the benzene ring). These inter­actions lead to the formation of zigzag ribbons which run along c and are approximately parallel to (110).

Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.

Author Summary

Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission.

Background

Intrinsically fluorescent glucose derived carbon nanospheres (CSP) efficiently enter mammalian cells and also cross the blood brain barrier (BBB). However, the mechanistic details of CSP entry inside mammalian cells and its specificity are not known.

Results

In this report, the biochemical and cellular mechanism of CSP entry into the living cell have been investigated. By employing confocal imaging we show that CSP entry into the mammalian cells is an ATP-dependent clathrin mediated endocytosis process. Zeta potential studies suggest that it has a strong preference for cells which possess high levels of glucose transporters such as the glial cells, thereby enabling it to target individual organs/tissues such as the brain with increased specificity.

Conclusion

The endocytosis of Glucose derived CSP into mammalian cells is an ATP dependent process mediated by clathrin coated pits. CSPs utilize the surface functional groups to target cells containing glucose transporters on its membrane thereby implicating a potential application for specific targeting of the brain or cancer cells.

Objective

To standardize the characterization of motor evoked potential (MEP) and cortical silent period (CSP) recordings elicited with transcranial magnetic stimulation (TMS).

Methods

A computer-based, automated-parameterization program (APP) was developed and tested which provides a comprehensive set of electromyography (EMG) magnitude and temporal measures. The APP was tested using MEP, CSP, and isolated CSP (iCSP) TMS stimulus-response data from a healthy adult population (N = 13).

Results

The APP had the highest internal reliability (Cronbach’s alpha = .98) for CSP offset time compared with two prominent automated methods. The immediate post-CSP EMG recovery level was 49% higher than the pre-TMS EMG level. MEP size (peak amplitude, mean amplitude, peak-to-peak amplitude, and area) correlated higher with effective E-field (Eeff) than other intensity measures (r ≈ 0.5 vs. r ≈ 0.3) suggesting that Eeff is better suited for standardizing MEP stimulus-response relationships.

Conclusions

The APP successfully characterized individual and mean epochs containing MEP, CSP, and iCSP responses. The APP provided common signal and temporal measures consistent with previous studies and novel additional parameters. Significance: With the use of the APP modeling method and the Eeff, a standard approach for the analysis and reporting of MEP-CSP complex and iCSP measurements is achievable.

When exponentially growing Vibrio cholerae cells were shifted from 37°C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15°C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspAVC) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspAVC and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspAVC and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37°C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5′ untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.

Incorporating receptor flexibility is considered crucial for improvement of docking-based virtual screening. With an abundance of crystallographic structures freely available, docking with multiple crystal structures is believed to be a practical approach to cope with protein flexibility. Here we describe a successful application of the docking of multiple structures to discover novel and potent Chk1 inhibitors. Forty-six Chk1 structures were first compared in single structure docking by predicting the binding mode and recovering known ligands. Combinations of different protein structures were then compared by recovery of known ligands and an optimal ensemble of Chk1 structures were selected. The chosen structures were used in the virtual screening of over 60,000 diverse compounds for Chk1 inhibitors. Six novel compounds ranked at the top of the hits list were tested experimentally and two of these compounds inhibited Chk1 activity–the best with an IC50 value of 9.6 μM. Further study indicated that achieving a better enrichment and identifying more diverse compounds was more likely using multiple structures than using only a single structure even when protein structures were randomly selected. Taking into account conformational energy difference did not help to improve enrichment in the top ranked list.

In this study, an experiment was designed to optimize the synthesis of seleno-Capparis spionosa L. polysaccharide (Se-CSPS) by response surface methodology. Three independent variables (reaction time, reaction temperature and ratio of Na2SeO3 to CSPS) were tested. Furthermore, the thermal stability, particle size, shape and cytotoxic activity of Se-CSPS in vitro were investigated. The optimum reaction conditions were obtained shown as follows: reaction time 7.5 h, reaction temperature 71 °C, and ratio of Na2SeO3 to CSPS 0.9 g/g. Under these conditions, the Se content in Se-CSPS reached 5.547 mg/g, which was close to the predicted value (5.518 mg/g) by the model. The thermal stability, particle size and shape of Se-CSPS were significantly different from those of CSPS. Additionally, a MTT assay indicated that the Se-CSPS could inhibit the proliferation of human gastric cancer SGC-7901 cells in a dose-dependent manner.

In the title compound, C26H43N7O, each piperazine ring adopts a chair conformation. Two 1-methyl­piperazine rings bond to one Csp 3 of the pyrrole ring via the piperazine N atoms, while the third one links to the N atom of the indolin-2-one unit through a flexible ethyl group with an almost syn conformation. In the crystal, mol­ecules are connected through methyl­ene–carbonyl C—H⋯O inter­actions into an infinite chain along the c axis. The almost parallel arrays are stacked, forming a three-dimensional framework.

Two cold shock genes, cspL and cspP, have been cloned from two Lactobacillus plantarum strains. These genes, which are nonallelic, were present in all strains tested. The genes encode 66-amino-acid polypeptides related to each other and to the cold shock Csp family. Transcription of cspP rendered a single mRNA, while two cspL mRNAs were found with common 5' ends. The amounts of these transcripts increased moderately upon exposure of the cultures to cold.

A homolog of the major eubacterial cold shock gene cspA was identified in Sinorhizobium meliloti RM1021 by luxAB reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of the Escherichia coli ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sized cspA-hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encoding cspA alone and a 1,000-nt transcript encoding cspA-ORF2-rpsU. The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of the cspA-ORF2-rpsU locus had −10 and −35 elements similar to the E. coli ς70 consensus promoter and, like the cspA locus of E. coli, included an AT-rich region upstream of the −35 hexamer. The promoter of the S. meliloti cspA locus was found to impart cold shock-induced mRNA accumulation. In addition, the 5′-untranslated region (5′ UTR) was found to increase the fold induction of cspA transcripts after cold shock and depressed the level of luxAB mRNA prior to cold shock, another feature similar to cspA regulation in E. coli. No “cold box” was identified upstream of the S. meliloti cspA gene, however, and there was no other obvious sequence identity between the S. meliloti 5′ UTR and that of E. coli. DNA hybridization analysis indicated that outside the cspA-ORF2-rpsU cold shock locus there are several additional cspA-like genes and a second rpsU homolog.