- Volume 148, Issue 10, 2002

PvdS is an alternative sigma factor regulated by the global iron regulator Fur. It has been demonstrated that PvdS plays a role in the iron-dependent regulation of exotoxin A (ETA) in Pseudomonas aeruginosa strain PAO1. The goals of this research were to determine if pvdS was transcribed by the bacteria in the chronic lung infections associated with cystic fibrosis (CF) and to determine how PvdS interacts with the regAB promoters of the hyper-toxigenic strain PA103. It was found that pvdS is transcribed in the lungs of patients with CF and that it appears to be involved with the regulation of toxA in this environment. This correlated with the finding that in strain PA103, a mutation in pvdS reduced ETA activity while the same mutation in strain PAO1 abrogated ETA production. It was also shown that in strain PA103, pvdS was absolutely required for activation of the regAB P2 promoter. The effect of PvdS on the P2 promoter may be direct or indirect; however, in support of a direct role, an eight-out-of-nine base-pair match to the consensus sequence for PvdS binding was identified at the transcriptional start site for the P2 promoter. The effect of PvdS on the PA103 regAB P1 promoter under aerobic growth conditions was also examined. The results show that PvdS does modulate the expression from this promoter but that both the regAB operon and PvdS are required for optimal P1 promoter activity. These studies demonstrate that the alternative sigma factor PvdS acts as a regulator of ETA expression in P. aeruginosa strain PA103 through the regAB operon and that PvdS is expressed in lung infections associated with CF.

Pseudomonas aeruginosa PAO1 was found to exhibit several remarkable physiological responses to oxidative stress upon its growth in a computer-controlled suspension culture. First, it strongly reduced the transfer rate of oxygen from the gas into the liquid phase, causing oxygen-limited or microaerophilic conditions in the culture after a short period of cultivation, even at high aeration rates with pure oxygen. Second, PAO1 that was previously classified as ’non-mucoid’ formed a clear polysaccharide capsule on the cell surface (mucoid phenotype) under oxidative-stress conditions. Third, the strain showed a reduced growth rate and a longer lag phase under high oxygen tension. Finally, P. aeruginosa PAO1 released a high amount of proteins into the culture broth. The release of some virulence factors by PAO1, such as elastase, was significantly enhanced or only occurred under microaerobic conditions (i.e. dissolved oxygen tension value around 1% of air saturation). Hence, it is concluded that P. aeruginosa PAO1 prefers microaerobic conditions for growth and for the formation of some of its virulence factors. PAO1 can create such growth conditions by at least two mechanisms: (i) blockage of the transfer of oxygen and (ii) formation of a polysaccharide capsule on the cell surface. It is postulated that the blockage of oxygen transfer may play an important role in the defence of this pathogen against reactive oxygen intermediates.

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the γ-subgroup of the Proteobacteria. This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the β-subgroup of the Proteobacteria. Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O2 and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3·4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O2 levels, results which were corroborated by GUS (β-glucuronidase) assays using a transcriptional nifL::gusA fusion. N2 fixation was abolished in a NifLA− and a NifA− mutant, wild-type fixation being restored by nifLA in trans. The NifLA− mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O2, suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O2 and combined nitrogen as NifL in bacteria of the γ-Proteobacteria.

The Min system of Escherichia coli directs cell division to the mid-cell by a mechanism that involves the dynamic localization of all of its three constituent proteins, MinC, MinD and MinE. Both the Min system and the nucleoid regulate cell division negatively and strains of E. coli lacking a functional Min system can divide at nucleoid-free cell poles in addition to the nucleoid-free region between newly segregated nucleoids. Interestingly, E. coli strains with a defective Min system have disturbed nucleoid segregation and the cause for this disturbance is not known. It is reported here that growth conditions promoting a higher frequency of polar divisions also lead to a more pronounced disturbance in nucleoid segregation. In strains with an intact Min system, expression of MinE, but not of MinD, from an inducible promoter was followed by impaired nucleoid segregation. These results suggest that the disturbed nucleoid segregation in min mutants is not caused by polar divisions per se, nor by impaired resolution of chromosome dimers in min mutants, leaving open the possibility that the Min system has a direct effect on nucleoid segregation. It is also shown how the disturbed nucleoid segregation can explain in part the unexpected finding that the clear majority of cells in min mutant populations contain 2 n (n=0, 1, 2…) origins of replication.

Using a continuous culture of Bacillus methanolicus MGA3 limited by 100 mM methanol in the feed and growing at a dilution rate D=0·25 h−1, transients in dissolved methanol were studied to determine the effects of methanol toxicity and the pathway of methanol dissimilation to CO2. Steady-state cultures were disturbed by pulses of methanol resulting in a rapid change in concentration of 6·4–12·8 mM. B. methanolicus MGA3 responded to a sudden increase in available methanol by a transient decline in the biomass concentration in the reactor. In most cases the culture returned to steady state between 4 and 12 h after pulse addition. However, at a methanol pulse of 12·8 mM, complete biomass washout occurred and the culture did not return to steady state. Integrating the response curves of the dry biomass concentration over a 12 h time period showed that a methanol pulse can cause an average transient decline in the biomass yield of up to 22%. 13C NMR experiments using labelled methanol indicated that the transient partial or complete biomass washout was probably caused by toxic accumulation of formaldehyde in the culture. These experiments also showed accumulation of formate, indicating that B. methanolicus possesses formaldehyde dehydrogenase and formate dehydrogenase activity resulting in a methanol dissimilation pathway via formate to CO2. Studies using isotope-ratio mass spectrometry provided further evidence of a methanol dissimilation pathway via formate. B. methanolicus MGA3, growing continuously under methanol limitation, consumed added formate at a rate of approximately 0·85 mmol l−1 h−1. Furthermore, significant accumulation of 13CO2 in the reactor exhaust gas was measured in response to a pulse addition of [13C]formic acid to the bioreactor. This indicates that B. methanolicus dissimilates methanol carbon to CO2 in order to detoxify formaldehyde by both a linear pathway to formate and a cyclic mechanism as part of the RuMP pathway.

A Staphylococcus aureus gene originally identified by signature-tagged mutagenesis as being required for virulence was cloned, sequenced and named svrA. Hydropathy profiles revealed that SvrA is likely to be membrane associated, having two regions with six membrane-spanning domains, the regions separated by an extended hydrophilic loop. When compared with the wild-type strain, an svrA mutant expressed greatly reduced amounts of α-, β- and δ-toxins and an increased amount of protein A. Toxin production by the mutant strain was restored to wild-type levels when complemented with a plasmid expressing the svrA gene. Northern hybridization with probes specific for hla (encoding α-toxin) and spa (encoding protein A) showed that the svrA mutant strain was affected in the transcription of these genes. svrA mRNA was present in wild-type and agr strains, but agr mRNA and RNAIII were absent in the svrA mutant strain. Virulence studies suggested that the attenuation of the svrA mutant was probably due to its direct or indirect effect on the agr regulon. These results indicate that svrA is required for the expression of agr and RNAIII transcripts and is therefore a new component of the agr regulatory network controlling virulence gene expression in S. aureus.

Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lens-shaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.

The Sca permease in the oral bacterium Streptococcus gordonii is a member of a family of ATP-binding cassette (ABC)-type transporters for manganese (Mn2+) and related cations that are associated with streptococcal virulence in a number of infection models. Since Mn2+ has a protective function against oxidative damage in a variety of bacteria, we have investigated the role of Sca permease in oxidative stress tolerance in Streptococcus gordonii. A single Mn2+-dependent superoxide dismutase (SOD), encoded by sodA, is expressed by S. gordonii and was >10-fold up-regulated under oxidative stress conditions. Inactivation of sodA resulted in increased susceptibility of S. gordonii cells to growth inhibition by dioxygen (O2), and to killing by paraquat (a superoxide anion generator) and by hydrogen peroxide (H2O2). Expression of thiol peroxidase, encoded by the tpx gene located immediately downstream of the scaCBA operon, was also up-regulated under oxidative conditions. Inactivation of tpx led to increased susceptibility of cells to H2O2, but not to O2 or paraquat. In low-Mn2+ medium (0·01 μM Mn2+) sodA and tpx genes were transcriptionally down-regulated, SOD activity was reduced and cells were more sensitive to growth inhibition by O2. A Sca permease-deficient (scaC) mutant showed further reduced SOD activity and hypersensitivity to O2 in medium containing <0·1 μM Mn2+. These results demonstrate that the Sca (Mn2+) permease in S. gordonii is essential for protection against oxidative stress.

In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheA-activating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

Among the few regulatory proteins encoded by Mycoplasma pneumoniae is HPr kinase/phosphatase (HPrK/P), the key regulator of carbon metabolism in low-GC Gram-positive bacteria. The corresponding gene, hprK, and the gene encoding the target protein HPr, ptsH, were overexpressed. In vitro analysis of the purified proteins confirmed ATP-dependent phosphorylation of HPr by HPrK/P. In contrast to HPrK/P of Bacillus subtilis, which is by default a phosphatase and needs high ATP concentrations for kinase activity, the M. pneumoniae enzyme exhibits kinase activity at very low ATP concentrations and depends on Pi for phosphatase activity. This inverted control of enzymic activity may result from the adaptation to very different ecological niches. While the standard activities of HPrK/P from M. pneumoniae and other Gram-positive bacteria differ, they are both modulated by the concentration of ATP, Pi and glycolytic intermediates. Site-directed mutagenesis of a potential ATP-binding site and of the HPrK/P signature sequence resulted in four different activity classes: (i) inactive proteins, (ii) enzymes with reduced kinase and phosphatase activities, (iii) enzymes that had lost phosphatase, but not kinase activity, and (iv) enzymes that exhibited increased phosphatase activity.

Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid Δ1-dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the Δ1-dehydrogenation of 4-androstene-3,17-dione (AD) and 9α-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35% identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid Δ1-dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

Slr1295 (and Slr0513) in the cyanobacterium Synechocystis sp. PCC 6803 has amino acid similarity to the bacterial FbpA protein family and also to IdiA of Synechococcus PCC 6301/PCC 7942. To determine whether Slr1295 is the periplasm-located component of an iron transporter, or has a function in protecting photosystem (PS) II, subcellular localization and Δslr1295 mutant characterization studies were performed. Localization of Slr1295 provided evidence that it has an intracellular function, since virtually no Slr1295 was detected in the soluble protein fraction of the periplasm or in the cytoplasmic membrane. Characterization of a Δslr1295 Synechocystis PCC 6803 mutant indicated that PS II is more susceptible to inactivation in the mutant than in the wild-type (WT). Under mild iron limitation, modification of PS I to the PS I–IsiA complex is more advanced in the Δslr1295 mutant, indicating that iron deficiency leads more rapidly to changes in the photosynthetic apparatus in the mutant than in the WT. Biochemical fractionation procedures provide evidence that Slr1295 co-purifies with PS II. These results suggest a function of Slr1295 that is comparable to the function of IdiA in Synechococcus PCC 6301/PCC 7942 being a protein that protects PS II under iron limitation in an as yet unknown way.

Single-stranded-DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in bacteria, archaea and eukarya. This paper reports the identification and characterization of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and Thermus aquaticus. These proteins (TthSSB and TaqSSB), in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers, and each monomer contains two ssDNA-binding domains with a conserved OB (oligonucleotide/oligosaccharide-binding) fold, as deduced from the sequence analysis. The N-terminal domain is located in the region from amino acid 1 to 123 and the C-terminal domain is located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively. Purified TthSSB or TaqSSB binds only to ssDNA and with high affinity. The binding site size for TaqSSB and TthSSB protein corresponds to 30–35 nucleotides. It is concluded that the SSBs of thermophilic and mesophilic bacteria, archaea and eukarya share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

The coumarin antibiotic coumermycin A1 contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A1 biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A1 production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A1, respectively. C-methylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the non-methylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A1 in the wild-type.

Since the activity of drugs against Mycobacterium tuberculosis grown in microbiological culture can differ from their activity against bacteria present in infected tissues, compounds with optimal activity against in vivo phenotypes may be overlooked in drug-discovery programmes that rely on in vitro screens. The authors have investigated the use of an ex vivo cell-culture model to assess the action of drugs on M. tuberculosis in an environment resembling that encountered during infection. Mycobacterial viability in the ex vivo model was shown to be regulated by the cell-mediated immune system, with growth inhibited by CD4+ T cells at an early stage of infection in BCG-vaccinated mice, and at a later stage after infection in naive mice. Screening of drugs in the ex vivo model demonstrated a window of pyrazinamide susceptibility that coincides with the onset of the T-cell-mediated immune response in naive or vaccinated mice. It is proposed that pyrazinamide acts on a population of bacteria that are exposed to an acidic environment as a result of immune activation. Clinically, administration of pyrazinamide during the initial phase of treatment reduces the risk of relapse after 6 months, suggesting that the early pyrazinamide-susceptible population may contribute to the later pool of mycobacteria that persist during prolonged chemotherapy.

The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14α-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0·2 and 0·3 μM, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1·2 and 36·5 μM, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K d values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor ΔCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.

Although most mycobacterial lipids are thought to be associated with the cell envelope, the authors previously observed substantial deposits of intracellular lipophilic material. A Nile-red-based cytological assay was used to determine factors which affect the presence and natural history of intracellular lipophilic inclusions (ILIs) in Mycobacterium smegmatis. Development of ILIs was associated with stationary-phase cultures in broth and with aged (6 days) colonies on agar. Using variants of Youmans’ defined medium, the frequency and size of ILIs was observed to be minimal in carbon-poor medium. ILIs were observed to form within 15 min after provision of fatty acids to the medium and after a period of several days in nitrogen-poor medium. Analysis of the non-polar lipid extracts of ILI-rich and -poor preparations indicated that the triacylglycerols (TAGs) were a major component of the inclusions. The acyl substituents of the TAGs varied according to whether they were formed in Middlebrook 7H9 broth, in low-nitrogen Youmans’ broth or rapidly after oleic acid supplementation of Youmans’ broth. These studies support a storage function for TAGs in mycobacteria in addition to their previously suggested occurrence as components of the cell envelope. To assess a possible role for ILIs in Mycobacterium tuberculosis, a combined acid-fast (Auramine)/Nile red assay was applied to heavily positive sputum samples from patients with tuberculosis. Strong intracellular Nile red signals were obtained from acid-fast cells, indicating that ILI occur in M. tuberculosis in vivo. This may reflect a distinct physiological state of these cells, which it has not been possible to reproduce in vitro. These findings indicate that the uptake of long-chain fatty acids and TAG biosynthetic and degradative pathways are important aspects of mycobacterial lipid metabolism, meriting further investigation.

Previous studies examining the expression of adhesion and integrin molecules on CD4 T lymphocytes generated in response to virulent Mycobacterium tuberculosis infection revealed that certain inbred mouse strains susceptible to breakdown of chronic disease and subsequent reactivation had poor expression of these molecules, which might underlie their inability to adequately focus into lung tissues and mediate protection. The current study examines the possibility that prior vaccination with BCG, or a prior tuberculosis infection, would overcome this deficiency. It was found, however, that this was not the case. Whereas both resistant (C57BL/6) and susceptible (DBA/2, CBA/J) strains were equally well protected in the spleen after intravenous challenge, the latter strains were poorly protected in the lungs regardless of whether the challenge was given by the intravenous or aerosol route. Again, this was associated with poor up-regulation of adhesion and integrin molecules and with histological evidence in memory immune animals of a reduced and delayed influx of T lymphocytes into the lungs.