RESULTS:

Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs) composed of 25–50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species named here as cas1B cas5 and cas6 and also revealed a certain number of spacers that have homology with extant genes most frequently derived from phages but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements and hypothesize that they provide the cell immunity against phage infection and more generally foreign DNA expression by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

In this report it is shown that the rolling circle replicon pG+host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria it is shown that IS1223 and IS1201 transpose in L. bulgaricus.

Summary: A 7 kb DNA fragment was cloned from Lactobacillus sakei which contains the IdhL gene encoding the l(+)-lactate dehydrogenase (l-LDH). Analysis of the DNA sequence Northern experiments and primer extension experiments showed that IdhL is transcribed from a single promoter leading to a monocistronic 1·15 kb mRNA which yields the l-LDH. A stable mutant was constructed by chromosomal integration of a chloramphenicol cassette into IdhL by a double-crossover event. Both l- and d-lactate were produced by the wild-type strain whereas only residual amounts of both isomers were produced by the mutant. This demonstrates that L. sakei possesses an l-LDH producing l-lactate and a lactate racemase able to transform it to d-lactate but is devoid of d-LDH activity. Moreover the ability to degrade l-lactate present in the medium that was observed with the mutant strain grown aerobically suggests that an l-lactate oxidase activity is also present in L. sakei.

SUMMARY: Transcription of a new catabolic operon in Bacillus subtilis involved in the late stages of galacturonic acid utilization has been studied. The operon consists of four genes: kdgR encoding the putative regulator protein; kdgK encoding 2-keto-3-deoxyg I uconate kinase; kdgA encoding 2-keto-3-deoxyg luconate-6-phosphate aldolase; and kdg encoding a transporter. These four genes are organized in one transcriptional unit and map at 198" of the B. subtik chromosome. Primer extension experiments and Northern blot analysis show that an active σ-dependent promoter precedes kdgR and transcription is terminated at the putative pindependent terminator downstream of kdgr. The operon is negatively regulated by the kdgR and ccpA gene products which belong t o the Lac1 family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove respectively the effects of galacturonate and glucose on the transcription of this operon.

The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275° on the genetic map was sequenced. The strategy applied based on use of yeast artificial chromosomes and multiplex Long Accurate PCR proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (Hi) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20 28 20 15 and 17 respectively. In the sequenced region a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140° in the chromosome was also observed.

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component sytems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments which clearly originated from response regulator genes were amplified from Escherichia coli Agrobacterium tumefaciens Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes which do not seem to be related to any of the already characterized genes was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.

Within the Bacillus subtilis genome sequencing project the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region between the kdg operon and the attachment site of the SPβ prophage. A two-step strategy was used for the sequencing. In the first step total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000 which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs) 19 of which encode unknown proteins. Seven genetic loci already mapped in this region xpt metB ilvA ilvD thyB dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA the first bacterial homologue of chalcone synthase; exol a 5′ to 3′ exonuclease similar to that of DNA polymerase I of Escherichia coli; and bsaA a stress-response-associated protein. The protein encoded by ypIP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.

The standard strategies of genome sequencing based on λ-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli . One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104 109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest previously sequenced contigs (13695 and 20303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning as well as for larger inserts of eukaryotic origin cloned ir YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region 57 have no clear putative function from their homology to proteins in the databases.

The dnaD gene of Bacillus subtilis was identified within a 104 kb DNA segment cloned into a yeast artificial chromosome. The nucleotide sequence of the wild type and dnaD23 mutant genes were determined. dnaD is predicted to encode a protein of 232 amino acids with no similarity to proteins in the data banks.