- Volume 143, Issue 3, 1997

asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an Escherichia coli δasd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-β-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal δasd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.

To examine whether mating can occur within as well as between clones of Trypanosoma brucei, we transformed three T. brucei subspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter- and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intraas well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase, THT1 glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.

Mycobacterium paratuberculosis promoter-containing clones were isolated from a genomic DNA library constructed in the transcriptional-translational fusion vector pYUB76. The promoter-containing DNA fragments were identified in the surrogate host Mycobacterium smegmatis by expression of the promoterless lacZ reporter gene of pYUB76. The expression signals exhibited a wide range of strengths, as indicated by their corresponding β-galactosidase activities. Eight clones were sequenced and characterized further. Predicted open reading frames and codon usage were identified by computer analysis. Database searching for related sequences using the BLAST method revealed no homologies. Transcriptional activity was measured by slot-blot hybridization with steady-state RNA isolated from lacZ + M. smegmatis clones. Primer extension analysis identified the transcription start sites within the cloned fragments. The promoter regions characterized in this study were used to establish a consensus promoter sequence for M. paratuberculosis. M. paratuberculosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centred approximately 35 and 10 bp upstream from the transcription startpoints do not correspond to the consensus hexanucleotides of Escherichia coli promoters.

The gene coding for the Mycobacterium tuberculosis homologue of LexA has been cloned and sequenced. Amino acids required for autocatalytic cleavage are conserved, whereas those important for specific DNA binding are not, when compared with Escherichia coli LexA. The transcriptional start site was mapped and a DNA sequence motif was identified which resembled the consensus Cheo box sequence involved in the regulation of DNA-damage-inducible genes in Bacillus subtilis. The M. tuberculosis LexA protein was overexpressed in E. coli and purified by means of a His tag. The purifed LexA was shown to bind to the Cheo box sequence found upstream of its own gene.

The Bacillus subtilis L-arabinose metabolic genes araA, araB and araD, encoding L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate 4-epimerase, respectively, have been cloned previously and the products of araB and araD were shown to be functionally homologous to their Escherichia coli counterparts by complementation experiments. Here we report that araA, araB and araD, whose inactivation leads to an Ara- phenotype, are the first three ORFs of a nine cistron transcriptional unit with a total length of 11 kb. This operon, called ara, is located at about 256 on the B. subtilis genetic map and contains six new genes named araL, araM, araN, araP, araQ and abfA. Expression of the ara operon is directed by a strong sA-like promoter identified within a 150 bp DNA fragment upstream from the translation start site of araA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP and araQ are homologous to bacterial components of binding-protein-dependent transport systems and abfA most probably encodes an a-L-arabinofuranosidase. The functions of araL and araM are unknown. An in vitro-constructed insertion-deletion mutation in the region downstream from araD allowed us to demonstrate that araL, araM, araN, araP, araQ and abfA are not essential for L-arabinose utilization. Studies with strains bearing transcriptional fusions of the operon to the E. coli lacZ gene revealed that expression from the ara promoter is induced by L-arabinose and repressed by glucose.

Members of the AAA-protein family are found in both prokaryotes and eukaryotes. These ATPases are involved in a number of diverse activities ranging from protein secretion to cell cycle control. This paper reports the functional analysis of the Bacillus subtilis ftsH gene, which encodes a member of this protein family. In cells containing reduced levels of a truncated FtsH protein cell growth was impaired under certain nutritional conditions. In a hypersaline environment FtsH was required in increased amounts for the cells' recovery from osmotic stress. In the absence of FtsH the abundance of several of the major penicillin-binding proteins (PBP2A and 2B) in the cytoplasmic membrane was affected. Lastly, it has been established that FtsH is required for entry into the developmental life cycle.

The Bacillus subtilis folate operon contains nine genes. The first six genes are involved in the biosynthesis of folic acid and tryptophan and have been characterized previously. The 3-region of the folate operon contains three additional ORFs: orf3, potentially encoding a DNA-binding protein of 68 amino acids, orf4, encoding a protein of 338 amino acids with homology to the Orf1 of the E. coli fis operon, and a putative lysyl-tRNA synthetase gene (lysS). Four transcripts were identified which encode the first two, eight or all nine proteins or only the last protein LysS. The folate operon contains two promoters, one upstream of the first gene and the second preceding lysS. Transcription of the entire folate operon starts 33 bp upstream of the ATG codon of pab, the first gene of the operon. The mtrB-encoded trp RNA-binding attenuation protein (TRAP) dramatically reduces the steady-state levels of the folate operon transcripts encoding the first eight and all nine proteins, but only has a relatively small effect on the steady-state level of the 2.1 kb transcript encoding the first two genes of the operon, pab and trpG. In addition, transcription of the folate operon is regulated in a growth-phase-dependent manner. Transcripts were present in very low levels after mid-exponential phase, but were dramatically increased directly after transfer of the cells to fresh medium. These results indicate that transcription of the folate operon is regulated by TRAP and also depends on the growth phase of the culture.

The marine purple non-sulphur bacterium Rhodovulum euryhalinum strain DSM 4868 reduced dimethyl sulphoxide (DMSO) to dimethyl sulphide (DMS) chemotrophically with sulphide as electron donor. The oxidation of sulphide was correlated with the formation of polysulphides. R. euryhalinum reduced DMSO phototrophically with sulphide as well, but the amount of DMSO reduced in relation to sulphide oxidized was lower. The marine green sulphur bacterium Chlorobium vibrioforme strain DSM 8327 reduced DMSO to DMS phototrophically with sulphide and thiosulphate as electron donors. The extent of DMSO reduction was much less in the dark. Eight strains of purple sulphur bacteria - marine, brackish water and freshwater isolates - and another marine green sulphur bacterium showed a very weak capacity for DMSO reduction with sulphide or thiosulphate as electron donors in the light and dark, respectively.

In Escherichia coli, expression of certain genes and operons, including the fructose operon, is controlled by Cra, the pleiotropic catabolite repressor/activator protein formerly known as FruR. In this study we have demonstrated that cra mutant strains synthesize 10-fold less cAMP than isogenic wild-type strains, specifically when grown in fructose-containing minimal media. The glucose-specific IIA protein (IIAglc) of the phosphotransferase system, which activates adenylate cyclase when phosphorylated, is largely dephosphorylated in cra but not wild-type strains growing under these conditions. Dephosphorylation of IIAglcin cra strains apparently results from enhanced fructose operon transcription and fructose uptake. These conclusions were supported by showing that fructose-grown cra strains possess 2·5-fold higher fructose-1-phosphate kinase activity than fructose-grown wild-type strains. Moreover, artificially increasing fructose operon expression in cells transporting fructose dramatically decreased the activity of adenylate cyclase. The results establish that Cra indirectly regulates the activity of adenylate cyclase by controlling the expression of the fructose operon in cells growing with fructose as the sole carbon source.

In Bacillus subtilis the Pho regulon is controlled by a sensor and regulator protein pair, PhoR and PhoP, that respond to phosphate concentrations. To facilitate studies of the Pho regulon, a strain with an altered PhoR protein was isolated by in vitro mutagenesis. The mutation in this strain (phoR12) leads to the production of a PhoR sensor kinase that, unlike the wild-type, is functionally active in phosphate-replete conditions. The lesion in phoR12 was shown to be a single base change that results in an Arg to Ser substitution in a region of PhoR that is highly conserved in histidine sensor kinases. While a phoR-negative mutant was unable to induce the synthesis of cell wall teichuronic acid under phosphate-limited conditions, the phoR12 mutant showed a relative increase in teichuronic acid and a decrease in teichoic acid, even under phosphate-replete conditions. The latter suggests that some or all of the genes required for teichuronic acid synthesis are members of the Pho regulon.

Twenty-three of the most prominent spots which are visible on two-dimensional (2-D) protein gels of Bacillus subtilis crude extracts were selected as marker spots for the construction of a 2-D protein index. N-terminal sequencing of the corresponding proteins resulted in the identification of enzymes involved in glycolysis, TCA cycle, pentose phosphate cycle, amino acid metabolism, nucleotide biosynthesis and translation. Using computer analysis of the 2-D protein gels, most of these metabolic enzymes were found to be synthesized at a reduced rate after different stresses and glucose starvation. Such an approach permits a rapid and global evaluation of the regulation of different branches of metabolism in response to various physiological conditions.

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of at least 42 general stress proteins absolutely required the alternative sigma factor sGB. However, at least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stress-inducible in a sigB mutant. Such a detailed analysis also permitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response. The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon. Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specific proteins is shown and discussed.

The outer-membrane (OM) permeability of the psychrotrophic bacterium Pseudomonas fluorescens strain MFO for the -lactam mezlocillin is increased at the optimum growth temperature (28 C) compared to low growth temperatures (8 C). In an attempt to explain this phenomenon, OM protein content was studied in cultures grown at both temperatures. No significant difference in proportion or composition was found, suggesting that a change in the structure and function of porins could be responsible for the differential permeability. The major OM protein OprF of two psychrotrophic P. fluorescens strains, MFO and OE 28.3, was purified from cultures grown at 8 C and 28 C in order to reincorporate them in solvent-free lipid bilayers. From cultures grown at the same temperature, OprF displayed very similar channel-forming properties for both strains. Decreasing the growth temperature induced a threefold reduction of the major conductance values (250270 pS in 1 M NaCl for 28 C cultures and 8090 pS in 1 M NaCl for 8 C cultures). The trypsin digestion kinetics showed a very different reactivity for these porins between cultures grown at 8 C and 28 C. This may indicate that the pore structure of OprF is modified depending on the growth temperature, as suggested by its functional behaviour.