- Volume 151, Issue 2, 2005

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a β-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Δfur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants.

The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 °C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 °C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane ΔpH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The ΔpH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the ΔpH component of the protonmotive force) and supplemented with glucose and Mg2+. The ΔpH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a ΔpH of 1·4 when butanol was added to a concentration of 1·5 % (w/v), while the wild-type ΔpH was reduced to 0·1. The ΔpH of both strains was completely dissipated with 1·75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the ΔpH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0·8 %) prevented this ΔpH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in ΔpH occurred more slowly than in the mutant, and sparing of the ΔpH by 0·8 % butanol was less pronounced. The authors interpret these data to mean that the ΔpH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.

Branched-chain fatty acids (BCFAs) typically constitute more than 90 % of the fatty acids of Listeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %) L. monocytogenes mutants (cld-1 and cld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917 was inserted into different genes of the branched-chain α-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, in which Tn917 was inserted into bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37 °C were stimulated by fatty acid precursors bypassing Bkd, 2-methylbutyrate (precursor for odd-numbered anteiso-fatty acids), isobutyrate (precursor for even-numbered iso-fatty acids) and isovalerate (precursor for odd-numbered iso-fatty acids). In contrast, the corresponding Bkd substrates, α-ketomethylvalerate, α-ketoisovalerate and α-ketoisocaproate, exhibited much poorer activity. At 26 °C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 °C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content of cld-2 from 33 % to 27 %, which may be close to the minimum BCFA requirement for L. monocytogenes. The transcription of bkd was enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors, cld-2 was able to increase its anteiso-C15 : 0 fatty acid content at 10 °C compared to 37 °C, which is the characteristic response of L. monocytogenes to low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis.

Streptococcus mutans is the primary odontopathogen present in supragingival plaque and causes the oral disease known as dental caries. Colonization of the oral cavity by S. mutans requires the bacteria to adhere to the tooth surface and occurs by both sucrose-dependent and -independent mechanisms. Sucrose-independent adhesion of S. mutans in vitro has been shown to involve an ORF (ORF0317) encoding a homologue (39 %) to LytR, a regulator of autolysin activity in Bacillus subtilis. The protein encoded by ORF0317, LytR, belongs to the LytR/CpsA/Psr protein family. This family has a putative role in cell-wall structural maintenance, possibly through autolysin regulation. Autolysins have also been shown to be important in surface adhesion in Lactococcus lactis and in the pathogenic properties of Streptococcus pneumoniae. To investigate the role of autolysins in the adhesion and pathogenesis of S. mutans, a LytR mutant was constructed. The mutant grows in long chains, which may indicate a defect in cell division. Further experiments with the mutant strain show increased autolytic activity, indicating that LytR attenuates S. mutans autolytic activity, possibly through regulation of the expression of autolytic enzymes. No defect in cell-to-surface adherence or biofilm growth was seen in the LytR mutant. However, a connection between cell growth phase and transcription of lytR was found.