Repulsion and metabolic switches in the collective behavior of bacterial colonies.

Bacteria inoculated on surfaces create colonies that spread out, forming patterns shaped by their mutual interactions. Here, by a combination of experiments and modeling, we address two striking phenomena observed when colonies spread out circularly, without dendritic instabilities. First, the velocity of spreading is generically found to decrease as levels of nutrients initially deposited on the surface increase. We demonstrate that the slowdown is due to phenomena of differentiation, leading to the coexistence of bacteria in different states of motility and we model their dynamics. Second, colonies spreading out from different inocula on the same surface are observed to merge or repel (halting at a finite distance), depending on experimental conditions. We identify the parameters that determine the fate of merging versus repulsion, and predict the profile of arrest in the cases of repulsion.

From protein sequence to function.

As genome sequences and protein structures are deciphered, we wish to predict their corresponding functions. Many functions cannot be told from from the sequence, however, although there has been progress in this quest for an impossible Grail. Furthermore, a structure and its corresponding sequence become most interesting when one knows the function. Inductive reasoning, based on the integration of biological and sequence knowledge, should enable sequence and functional data to be combined in a productive way.

Codon usage and lateral gene transfer in Bacillus subtilis.

Bacillus subtilis possesses three classes of genes, differing by their codon preference. One class corresponds to prophages or prophage-like elements, indicative of the existence of systematic lateral gene transfer in this organism. The nature of the selection pressure that operates on codon bias is beginning to be understood.

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.

Indigo: a World-Wide-Web review of genomes and gene functions.

The present article describes a genome database reviewing gene-related knowledge of two model bacteria, Bacillus subtilis and Escherichia coli. The database, Indigo, is open through the World-Wide Web (http://indigo.genetique.uvsq.fr). The concept used for organising the data, the concept of neighbourhood, allows one to explore the database content in an efficient although somewhat unusual way. Here, genes are related to each other by a variety of neighbourhoods, including proximity in the chromosome, phylogenetic kinship, participation in a common metabolic pathway, common presence in an article of the literature, or similar use of the genetic code. Several examples illustrate how this concept of neighbourhood permits one to review the available knowledge about a given gene or gene family, and elaborate unexpected, but revealing, analyses about gene functions.

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement.

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Escherichia coli molecular genetic map (1000Kbp):update.

The sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by kohara et al. (1987). One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome. The accuracy of the map is assessed, and it is corrected and updated where appropriate, A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al. (1987), ie. Pvull and EcoRV, The nucleotide environment of such missing sites was examined and, using an Artificial intelligence approach, it appears that the site for these enzymes is sensitive to context effects. Several genes of known position on the E. coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exists on the restriction map, in addition to the original seven identified by Kohara et al. We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map. Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col.

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg.

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

The PatB protein of Bacillus subtilis is a C-S-lyase.

The PatB protein of Bacillus subtilis had both cystathionine beta-lyase and cysteine desulfhydrase activities in vitro. The apparent K(m) value of the PatB protein for cystathionine was threefold higher than that of the MetC protein, the previously characterized cystathionine beta-lyase of B. subtilis. In the presence of cystathionine as sole sulfur source, the patB gene present on a multicopy plasmid restored the growth of a metC mutant. In addition, the patB metC double mutant was unable to grow in the presence of sulfate or cystine while the patB or metC single mutants grew similarly to the wild-type strains in the presence of the same sulfur sources. In a metC mutant, the PatB protein can replace the MetC enzyme in the methionine biosynthetic pathway.

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.

Positive regulation of the expression of the Escherichia coli pts operon. Identification of the regulatory regions.

The pts operon of Escherichia coli is composed of the ptsH, ptsI and crr genes coding for three proteins central to the phosphoenolpyruvate dependent phosphotransferase system (PTS), the HPr, enzyme I and EIIIGlc proteins, respectively. We previously showed that transcription from the promoter region located upstream from the pts operon is regulated by two control circuits, which can occur independently from each other. Transcription of the pts operon is (1) stimulated by the CAP-cAMP complex and (2) enhanced during growth on glucose, a PTS substrate. The DNA regions involved in regulation of the expression of the pts operon have been identified. Two promoters, P0 and P1, separated by 100 bp are located upstream from the pts operon. In these promoter regions, we identified two sequences showing similarity with the consensus of CAP-binding sites, CAPa located near P0 and CAPb located in the -35 region of P1. In vivo experiments showed that binding of CAP-cAMP at the CAPa site stimulates transcription from the P0 promoter. The binding sites of CAP-cAMP and/or RNA-polymerase on a DNA fragment containing both P0 and P1 promoters as well as both CAPa and CAPb sites were examined by the technique of DNase I footprinting. These in vitro experiments suggested that CAP-cAMP binding at the CAPb site might also play a role in regulation of the pts operon expression. In addition, we showed that the DNA region carrying the CAPa site is important for regulation by glucose. We finally propose that the expression of the pts operon is controlled by two alternative positive regulatory mechanisms, which are designed to allow activation of the pts operon under a great variety of growth conditions.

Two functional domains in adenylate cyclase of Escherichia coli.

To study the regulation of Escherichia coli adenylate cyclase, plasmids that carry part of the cya gene, as well as plasmids containing hybrid genes having part of cya fused to part of lacZ, were constructed. This allowed us to propose that the enzyme is made of at least two well-defined domains. The NH2 terminus carries the ATP leads to cAMP catalytic activity, whereas the COOH-terminal end corresponds to a regulatory polypeptide domain.

Identification of the Escherichia coli cya gene product as authentic adenylate cyclase.

The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase. Finally, the protein has been submitted to amino acid sequence analysis. It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .

Evidence for horizontal gene transfer in Escherichia coli speciation.

After extracting more than 780 identified Escherichia coli genes from available data libraries, we investigated the codon usage of the corresponding coding sequences and extended the study of gene classes, thus obtained, to the nature and intensity of short nucleotide sequence selection, related to constraints operating at the nucleotide level. Using Factorial Correspondence Analysis we found that three classes ought to be included in order to match all data now available. The first two classes, as known, encompass genes expressed either continuously at a high level, or at a low level and/or rarely; the third class consists of genes corresponding to surface elements of the cell, genes coming from mobile elements as well as genes resulting in a high fidelity of DNA replication. This suggests that bacterial strains cultivated in the laboratory have been fixed by specific use of antimutator genes that are horizontally exchanged.

Uneven distribution of GATC motifs in the Escherichia coli chromosome, its plasmids and its phages.

This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coli parasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.

Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP.

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.

Identification of yrrU as the methylthioadenosine nucleosidase gene in Bacillus subtilis.

Taking trimethoprim as the selective agent in the presence of thymine, we adapted to Bacillus subtilis a selection procedure depending on the peculiar organisation of the one-carbon metabolism. The corresponding pathways couple synthesis of thymine to tetrahydrofolate consumption as a substrate of the reaction mediated by thymidylate synthase, instead of being a co-enzyme as in the other reactions transferring one-carbon groups. Mutants obtained are thymidylate synthase deficient, and therefore auxotrophic for thymine. This provides positive selection in a first step for gene replacement by a thymidylate synthase cassette, and subsequently against its presence. For systematic recombination of mutations constructed in vitro, we used the property of B. subtilis to grow at high temperature, noting that the thyB gene product is inactive at 46 degrees C, while the product of thyA remains active at this temperature. As the first step, we built up a recipient thyA- background, deleting the gene by in situ recombination. This method was used to investigate the function of the yrrU gene, which is presumably involved in a sulfur recycling pathway associated with polyamine biosynthesis. We showed that yrrU codes for a protein recycling methylthioadenosine, probably a nucleosidase. In addition we observed that B. subtilis can use methylthioribose as a sulfur source, and that it is an efficient sulfur scavenger.

Using codon usage to predict genes origin: is the Escherichia coli outer membrane a patchwork of products from different genomes?

Analysis of the codon usage of genes coding for the structural components of the outer membrane in Escherichia coli, is consistent with the requirement for high expression of these genes. Because porins (which constitute the major protein component of the outer membrane), and LPS (which constitute the major outermost constituent of the outer membrane), are synthesized from genes displaying widely different codon usage, it is possible to investigate the origin of the outer membrane. The analysis predicts that the outer membrane might originate from a genome other than the genome coding for the major part of the cell. Such a special origin would explain in structural terms, the likely lethality of porins if they were inadvertently inserted within the inner membrane, giving rise to the Gram-negative bacterial type, having an envelope comprising two membranes, instead of a single cytoplasmic membrane and a murein sacculus.

Cloning and sequence of the Bordetella bronchiseptica adenylate cyclase-hemolysin-encoding gene: comparison with the Bordetella pertussis gene.

The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase-hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule.