Alessandra M. Albertini

Essential Bacillus subtilis genes

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions.

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.

Gene amplification in the lac region of E. coli

We have characterized strains of E. coli in which the lac region, together with varying amounts of surrounding DNA, is amplified 40 to 200 fold. The amplification events involve regions of 7 to 37 kb and result in a tandem array of repeated units. Restriction digest patterns of DNA from over 100 independent strains reveal that the amplified units are different in each case. Mechanisms of gene duplication and amplification, and the relationship of gene amplification in bacteria to that in eucaryotic cells, are considered.

Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168

A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.

A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases

The nucleotide (nt) sequence of 13.6 kb of the outG locus of Bacillus subtilis, which maps at approximately 155 degrees between the genetic markers nrdA and polC, was determined. One putative coding sequence was identified corresponding to a large polypeptide of 4427 amino acids (aa). Structural organization at the nt and aa sequence level and extensive similarities of the deduced product, especially to EryA, suggest that the locus is potentially responsible for the synthesis of a polyketide molecule. The locus has been renamed pksX. Comparison of the deduced product with known fatty acid and polyketide synthases (PKS) suggested the presence of beta-ketosynthase, dehydratase, beta-ketoreductase and acyl-carrier protein domains. Preliminary data obtained with deletion mutants indicate that pksX is not an essential gene.

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.

Sequence around the 159' region of the Bacillus subtilis genome : the pksX locus spans 33.6 kb

The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13,341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb.

The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13,341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.

Bacillus subtilis mutS mutL operon : identification, nucleotide sequence and mutagenesis

The Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150 degrees on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-lacZ transcriptional fusion. An increase in expression was observed during late exponential growth.