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A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase
ELSEVIER
FEMS Microbiology Letters 135( 1996) 123- 129
A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase Anjana Ray, Huw D. Williams Department
ofBiology.
Imperial
College of Science, Technology
and Medicine,
*
Prince Consort Road.
LondonSW7 2BB. UK
Received 11September 1995; revised 30 October 1995; accepted 3 I October 1995
Abstract Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N, N, N’, N’-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway’s terminal oxidase is a quinol oxidase. Keywords:
Pseudomonas
aeruginosa;
Respiration; Cytochrome; Cytochrome oxidase; Haem
1. Introduction Pseudomonas aeruginosa is found in a very wide range of ecological niche ranging from soils and water sources to the lungs of patients with cystic fibrosis and the sites of bum wound infections [l]. These diverse environments will certainly differ in the availability and concentration of dissolved 0, and so respiratory adaptability may play a key role in the growth and survival of P. aeruginosa. One branch of the P. aeruginosa respiratory chain has a terminal oxidase that is highly resistant to the classical respiratory inhibitor potassium cyanide [2,3]. The molecular nature of this cyanide-insensitive oxidase (CIO) has not been elucidated but it is of interest because of the ability of P. aeruginosa to synthesise HCN, at
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( 17I) 584 2056; E-mail: [email protected]. 0378-1097/96/$12.00 SD/
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concentrations of 200-300 PM. Such levels would inhibit the cytochrome c oxidase, cytochrome co, which terminates a second branch of the aerobic respiratory chain [2-41. A third terminal oxidase, a cytochrome baa,, has been purified from a strain of P. aeruginosa and it is sensitive to cyanide (Iso = 50 PM), although there is no evidence yet for this oxidase in the well-characterised PA0 strains [2,5]. We are interested in the specific roles played by the terminal oxidases of this bacterium in its physiological adaptation to different environments. To this end we have isolated mutants defective in the CIO and cloned the complementary structural genes [2] and their sequence and characterisation will be published elsewhere. We are also investigating mutants defective in the cytochrome co-terminated pathway, both to aid genetic and physiological studies of this cytochrome c oxidase and to allow us to probe in greater detail the properties of the CIO-terminated
Federation of European Microbiological Societies. All rights reserved
pathway. The presence of c-type cytochromes in a respiratory chain allows the artificial electron donor N, N, N’, N’-tetramethyl-p-phenylenediamine (TMPD) to be oxidised to form an indophenol blue compound. Organisms that are unable to oxidise TMPD or do so poorly contain little or no cytochrome c in their aerobic respiratory chains [6]. Therefore, oxidation of TMPD provides both a means of specifically assaying the electron flux to OZ through a cytochrome c oxidase and a useful screen for isolating mutants defective in these pathways [3,7]. We have isolated a number of TMPD oxidase-defective mutants of P. aeruginosa following transposon mutagenesis and it is the properties of one such mutant that we describe in this paper.
2. Materials
and methods
2. I. Bacterial strains, media and growth conditions Pseudomonas aeruginosa PA06049 met-901 1 amiE200 strA [8], referred to as the wild-type throughout this paper, was used for Tn5-7.51 mutagenesis. PA07720 is the ccm mutant strain derived by Tn 5-751 mutagenesis of PA06049, and PA0772 1 is the ccm mutant complemented with the pADD386-based recombinant replicon PARS. Escherichia coli ED8654 metB supE supF hsdM hsdR was transformed with pME9, a derivative of the IncP-I based-plasmid pME305, which carries Tn5751 and acts as its delivery vehicle [S]. P. aeruginosa was grown aerobically in Luria-Bertani (LB) agar and LB broth [9], nutrient agar and nutrient yeast broth (Nutrient broth supplemented with 0.5% yeast extract). The incubation temperature was usually 30°C for PA06049 and for strains carrying pME9. Incubation at 43°C (non-permissive temperature for pME9) was used to cure strains of the plasmid. E. coli strains were grown at 37°C in LB. Anaerobic growth of P. aeruginosa was on LB + 0.2% KNO, and incubation was at 30°C in an anaerobic jar (Gas Pak, Becton Dickinson Co.). As required, antibiotics were added at the following concentrations ( pg ml ’ ), for E. coli: tetracycline (Tc, 251, kanamycin (Km, 251, trimethoprim (Tp, IOO), ampicillin (Ap, 100) and streptomycin (Sm, 200); and for P. aeruginosa: carbenicillin (Cb, 500), Tc (1251, Km (300) and Tp (500).
2.2. Preparation
of membranes
Membranes were prepared as described previously [2] from cells grown in LB broth until late exponential phase (OD,,,,, = I .O>. 2.3. Transposon mutagenesis and TMPD oxidase-negatil,e mutants
screening
,fbr
P. aeruginosa PA06049 was mutagenised using the Tn5-derivative, Tn5-751 [8]. The E. coli strain ED8654/pME9 carries Tn5-751 on the mobilisable plasmid replicon pME9. This strain served as a donor strain for conjugation with PA06049. E. coli ED8654/pME9 was grown at 30°C in nutrient yeast broth containing Km and Tc, whereas the recipient PA06049 was grown at 43°C to reduce activity of its restriction system [8]. For mating 5 ml volumes of late exponential phase cultures of donor and recipient cultures were mixed, pelleted by centrifugation and resuspended in 0.1 ml of medium and spread onto a nutrient agar plate to allow conjugation. After 4 h at 30°C the cells from the conjugation plate were collected by resuspension in 2 ml of saline (0.9% (w/v) NaCI) and plated onto NA + Tp, Km and Sm, and incubated at 30°C for 2-3 days. Simultaneous selection for resistance to Tp and Km selects for the presence of Tn 5-751 and eliminates the possibility of isolating spontaneous mutants. Sm counter selects against the E. coli donor strain. About 30000 KmR TpR SmK exconjugants were screened for loss of cytochrome c oxidase activity. This test uses the ability of the cytochrome c oxidase pathway to oxidise the artificial electron donor TMPD to an indophenol blue compound. The test was done by replica plating the exconjugants onto filter paper and then spraying them with a 9 mM solution of TMPD. Colonies that failed to turn blue after 30 s were taken as potential cytochrome c oxidase-negative mutants. Putative mutants were grown at 43°C to promote loss of the donor plasmid pME9 and its loss was verified by plating on NA + Tc and NA + Cb. 2.4. In l~ilv cloning
of complementing
genes
P. aeruginosa chromosomal DNA capable of complementing the TMPD oxidase-negative phenotype was cloned using the in vivo cloning system of Darzins et al. [ IO,1 I], using the mini-D31 12 based
replicon pADD386. The pADD386based gene library was used to transduce the oxidase mutant to CbR and transductants were subsequently screened for recovery of the ability to oxidise TMPD as described above. 2.5. C_vtochrome analysis Analysis of the cytochrome content of cells and membranes, using reduced-minus-oxidised and carbon monoxide (CO)-difference spectra was carried out as previously described [2,12]. Haem staining of membrane proteins to analyse for proteins with covalently bound haem c first involved pretreatment of membrane samples to remove potential interference from b-type cytochromes as described by Goodhew et al. [ 131. The pellet was then dissolved in electrophoresis sample buffer before SDS-PAGE and gels were stained for haem containing proteins using tetramethylbenzidine (TMBZ) as described by Thomas et al. [ 141. 2.6. Meusurement
of respirator? activities
Measurement of substrate-dependent 0, uptake rates. cyanide titration of activities and cytochrome c oxidase activity was carried out as previously described [2]. Myxathiazol-insensitive respiration was determined as 0, uptake in the presence of 100 PM myxathiazol, a concentration known to inhibit electron transfer through bacterial cytochrome bc, complexes [ 15,211. TMPD oxidase activity was assayed in membranes by following the increase in absorbance at 520 nm because of oxidation of TMPD [3]. All enzyme activity data represent the mean values from two or three experiments and there was typically no more than 10% variation between experiments. Protein assays were carried out using the method of Markwell et al. [16].
ability to oxidise TMPD to an indophenol blue compound. Twelve Tn5-751 insertion mutants were found which failed to turn blue or showed only slight blue coloration within 30 s of TMPD treatment. TMPD oxidase-deficient mutants could result from a number of possible respiratory defects. These include mutation of the structural genes of an obligatory component of the cytochrome (’ oxidase-terminated pathway, such as those for a cytochrome c’ or the terminal oxidase itself. Alternatively, the phenotype could result from a defect in the synthesis or assembly of a haem prosthetic group or some other essential co-factor or from mutation of a regulatory gene. The properties of one of these TMPD oxidase mutants, PA07720. are the subject of the remdmder of this paper. The mutant was complemented using the in vivo cloning system for P. aeruginosa based upon bacteriophage D3112 [ 10.1 I]. Phagemid Iysates of the mini-D replicon pADD386 carrying randomly packaged fragments of the P. aeruginosa chromosome were used to transduce PA07720 to CbR and transductants were screened for acquisition of a TMPD oxidase-positive phenotype. One complementing clone. PARS, was isolated and chosen fat further study. Both PA06049 and the mutant PA07720 had similar growth rates in LB although the mutant grew to significantly lower final optical densities than the wild-type (I .3S and 0.87 for PA06049 and PA07720, respectively). However. the mutant PA07720 was unable to grow anaerobically with nitrate as its terminal electron acceptor. The complemented mutant strain PA0772 I had identical growth characteristics to the wild-type. The lower final growth yield of the mutant is consistent with the loss of a thermodynamically efficient respiratory pathway. The inability to grow anaarohically on nitrate containing medium indicates a pleiotropic respiratory defect, affecting both the aerobic and anaerobic respiratory chains. 3.2. Biochemicul
charucterisation
of the mutarlt
3. Results and discussion 3.1. Isolation and complementation of mutants of P. aeruginosa dqfectir-e in TMPD oxidase actiri? Approximately 30000 KmR TpR exconjugants of P. aeruginosa PA06049 were screened for their
The only clearly demonstrated cytochrome c oxidase of P. aeruginosa PA06049 is cytochrome co [3,17]. The activity of the cytochrome co-terminated respiratory chain was measured by determining TMPD and cytochrome c oxidase activities. As can be seen in Table 1. membrane preparations from the
126
A. Ray, H.D. Williams/ FEMS Microbiology Letters 135 (19961 123-129
Table 1 TMPD and cytochrome PA0772 1
c oxidase
activities
Strain
in membranes
of P. aeruginosa wild-type,
TMPD oxidase activity (relative level)
PA06049 PA07720 PA07721
0.98 (I) ND ’ (0) 2.35 (2.4)
ccm ccm/pARS
a Given in pmol TMPD oxidised minb Given in pmol cytochrome c oxidised ’ ND, no activity detectable.
L
3,.
I,
I,,
550 Uavdeqth
1
MN) (nmn)
and complemented
mutant
Cytochrome c oxidase activity b (relative level)
’ (mg protein)-‘. min- ’ (mg protein)- ’
I,.......
SW
PA07720
54.7 (1) 7.8 (0.14) 60.6 (I.1 I)
mutant PA07720 had no TMPD oxidase activity and only 14% of the cytochrome c oxidase activity of the wild-type. pAR5 complemented these activities back to wild-type or higher levels. These results were expected from the plate assay phenotypes. Fig. 1 shows difference spectra of membranes of the various strains. In reduced-minus-oxidised spectra PA06049 shows an cz-maximum at 552 nm due to c-type cytochromes and a prominent shoulder at 558 nm due to cytochromes b (Fig. 1A). In contrast, in the mutant PA07720, the 552 nm peak corresponding to cytochromes c is absent and instead there is a maximum at 558 nm due to b- and possible o-type cytochromes (Fig. 1B). Approximate quantification of these spectral signals indicates that
500
A
mutant
1
554 Wavekgth
6W (nml
Fig. I. Difference spectra of membranes from wild-type, ccrn mutant and complemented mutant strains of P. aeruginosu. Reduced-minus-oxidised (A, B and C) and carbon monoxide (CO)difference spectra (D, E and F) were recorded of membranes from PA06049 (A, D>; PA07720 ccm (B, E) and PA0772 1 ccm /PARS (C, F). The vertical bar represents a change in absorbance (A A) of 0.06 (A, B and C), 0.012 (D and E) and 0.02 (F). All samples were at a protein concentration of 2 mg ml- ’
there is at least 70% decrease in the levels of c-type cytochromes in the mutant, Spectra of whole-cell samples gave similar results (data not shown). In the complemented mutant the cytochrome c signal at 552 nm reappears (Fig. 1C) and the levels of both cytochromes b and c are about 2.5 times higher than in the wild-type. The effect of the mutation on CO-binding cytochromes, and hence potential terminal oxidases, was determined from CO-difference spectra. PA06049 shows an asymmetric trough with minima at 550 and 558 nm probably largely due to the CO-binding haems c and o/b of the terminal oxidase, cytochrome co (Fig. 1D) [3,17]. The COdifference spectrum of the mutant is strikingly different (Fig. IE). Its spectrum is dominated by a large increase in the levels of a CO-binding cytochrome b with a deep trough at 558 nm. This would be explained if the mutation in PA07720 directly or indirectly led to the loss of a terminal respiratory pathway and there was a compensatory increase in the levels of a second pathway containing a CO-binding haem b/u. Complementation with pAR5 led to recovery of the wild-type spectral features, although the levels of CO-binding cytochrome c and cytochrome b were higher than in the wild-type (Fig. 1F). To establish more clearly whether the mutant had a pleiotropic defect in c-type cytochromes, crude extracts and membranes of the various strains were analysed by haem staining of gels following SDSPAGE. Membranes from the wild-type contained multiple haem staining bands (Fig. 2, lane 1). In contrast, no haem c-staining bands were present in the mutant PA07720 (Fig. 2, lane 2) while complementation by pAR5 led to recovery of all bands (Fig. 2, lane 3). So the spectral and haem staining gel data
127
A. Ray, H.D. Williams/ FEMS Microbiology Letters 135 (1996) 123-129
found that in TMPD- mutants of Rhodobucter cupsulutus, defective in genes subsequently shown to have roles in biogenesis of cytochrome c, had less than 20% of the spectrally detectable cytochrome c compared to the wild-type [19]. The fact that b-type cytochromes are still produced in P. aerwginosrr PA07720 indicates that the mutation does not have a pleiotropic defect in protoporphyrin IX synthesis. It suggests that the mutant is unable to form mature c-type cytochromes. The inability of the mutant to grow anaerobically is consistent with a cytochrome c maturation defect as a cytochrome c containing pathway is required for nitrite respiration, which occurs during anaerobic growth with nitrate [20]1. Haem groups of c-type cytochromes are covalantly attached to polypeptides via two covalent bonds formed between thiol groups of the polypeptide ‘and the vinyl groups of protohaem IX. Haem lyases, the enzymes which catalyse this reaction, have been identified in mitochondria [21] but none have yet been identified in bacteria, although it is assumed that they exist [ 181. Several other genes withipossible roles in cytochrome c biogenesis or maturation have been identified in bacteria (for review see [IS]). Sequencing of the complementing gene in pAR5 would obviously give some clues to its function if its sequence was homologous to known cytochrome c biogenesis genes. It has recently been suggested that a standard nomenclature be used in naming genes involved in these processes, namely that, they be called ccm genes for cytochrome c maturation [22]. While we have not as yet identified a specific gene
Fig. 2. Haem staining of polypeptides from membranes of wildtype, ccm mutant and complemented mutant strains. Membrane proteins were separated by SDS-PAGE and stained for haem with TMBZ as described in Materials and methods. Approximately 200 pg of protein was loaded in each lane. Lane 1, PA06049; lane 2, PA07720 ccm; lane 3, PA07721 ccm/pARS. The molecular mass markers at the side of lane I are, from top to bottom: 68, 43, 29, 18 and 14 kDa.
show that most if not all of the c-type cytochromes have been lost in the TMPD oxidase mutant. From these results it is clear that the mutation PA07720 has a pleiotropic effect on all haem c containing proteins in the membranes of P. aeruginosa. This P. aeruginosa mutant is reminiscent of mutants isolated, using similar screening procedures, in a number of different bacteria which have been shown to have defects in cytochrome c biogenesis. Kranz
Table 2 Comparison
of the respiratory
Strain
PA06049 PA07720 ccm PA0772 I ccm / pAR5
activities
of P. aeruginosa wild-type,
NADH-dependent Oz uptake a (relative levels)
CIO activity b (relative levels)
102 (1) 576 (5.6) 400 (3.9)
35 (1) 527 (15.1) 164 (4.7)
ccm mutant and complemented CIO activity
strains Myxathiazol-insensitive respiration ’ (%b)
(%o)
Myxathiazol-insensitive respiration ’ (relative levels)
34 91 41
49 (1) 421 (8.6) ND f
48 73 ND
’
min- ’ (mg protein)- ’ 0, uptake in the presence of 1 mM KCN, given in nmol 0, mm’ (mg protein)-‘. ’ CIO activity as a percentage of total NADH-dependent 0, uptake. d NADH-dependent 0, uptake in the presence of 100 PM myxathiazol, given in nmol 0, min- ’ (mg protein)’ Myxathiazol-insensitive respiration as a percentage of total NADH-dependent 0, uptake. ’ ND, not determined. a Given in nmol 0,
b NADH-dependent
’
or its function, we feel justified for the reasons given in referring to the mutated gene as a ccm gene. TMPD oxidase mutants of P. aeruginnsa have been isolated previously following chemical mutagenesis. Matsushita et al. [3] described the characterisation of such a mutant which, although it had no observable changes in cytochrome composition, was instrumental in demonstrating the presence of cyanide-insensitive respiration in this bacterium. While Yang [7] isolated a TMPD- mutant which did have lowered cytochrome c levels, the function of its cyanide-insensitive respiratory chain was not investigated. A mutant with a pleiotropic defect in c-type cytochromes allowed us to look at the role of c-type cytochromes in cyanide-insensitive respiration. The cyanide-insensitive oxidase (CIO) has not been isolated and it is not clear where its pathway branches off the main respiratory chain and hence whether its immediate electron donor is cytochrome c or a quinol. In the ccm mutant, malate-, succinate-, glucose- and gluconate-dependent 0, uptake rates were little changed (data not shown). However, NADHdependent O? uptake was more than five-fold higher in the mutant (Table 2). The activity was slightly lower in the complemented mutant but remained four-fold greater than the wild-type. Fig. 3 shows cyanide titration curves of NADH-dependent O? uptake. The wild-type had the expected biphasic titration curve [2,3] which indicates the presence in the membranes of at least two terminal oxidases with differing sensitivities to cyanide, with the more resistant respiration being due to the cyanide-insensitive oxidase [2]. In contrast, the ccm mutant PA07720 shows a monophasic titration curve with all respiration being cyanide-insensitive. This indicates that the entire NADH-dependent O2 uptake of this mutant is due to increased activity of the (JO-terminated pathway that corresponds to a ISfold increase in CIO activity (Table 2). This result clearly shows that the ccm mutant, which has lost all c-type cytochromes, still has a fully functional CIO-terminated respiratory pathway. The loss of the cytochrome c oxidase pathway leads to a massive increase in CIO activity, possibly in an attempt to compensate for the loss of electron flux to 0, through an energetically efficient cytochrome c oxidase. Complementation of the ccm mutation led to recovery of the biphasic titration curve (Fig. 3) and similar proportion of respiration in
‘ool~ 80
1
\T
Cyanide Concentration (M) Fig. 3. The effect of KCN on NADH-dependent the membranes of P. rrerqirww polarographically 100%
activity
PA07720
strains.
as described in Materials values
are
cc,,? (0 ), PA0772
given
in
oxygen uptake of
Activities were measured Table
I L’UII/PARS
and methods. The 2.
PA06049
cm).
( A ).
this strain was cyanide-insensitive as in the wild-type. However, the actual levels of respiration were still much higher than in the wild-type and this seems due to greater activity of both the CIO and cyanidesensitive pathways (Table 2) although this was not correlated with an increase in cytochrome L’oxidase activity (Table 1). We determined the effect of myxathiazol, which inhibits electron transfer through the ubiquinone-cytochrome c oxidoreductase complex (cytochrome hi.,), on O? uptake. The ccnz mutant PA07720 had a more than eight-fold increase in myxathiazol-insensitive respiration that accounted for at least 70% of all respiratory activity in this strain. Therefore, the CIO terminates a myxathiazol-insensitive pathway and confirms our expectations of a terminal oxidase that is able to function without c-type cytochromes, that is, it is most probably a quinol oxidase. While the molecular nature of the CIO has not been established, it is interesting to note that in the ccm mutant, which showed a ISfold increase in CIO activity, the only distinct spectral signal becoming more prominent is that of CO-binding cytochromes h.
[ 101Darwin\.
Acknowledgements
A. and Casaadaban. M.J. (I 989) In viva cloning oi
P,iu~frk~~~~or~o.~ omr,~i~~o.sc~genes with Mini-D?
This research was supported by a grant from the SERC (GR/F82214) to H.D.W. We thank D. Haas for providing the TnS-751 mutagenesis system and A. Darzins for providing the mini-D-based in vivo cloning system.
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H.D. and Poole. R.K.
The cytoch;rotnc\ 01
~~~c,rohoc~rc,~ I’[‘.~tC,l,~i~l,r~,) NCIB 641X. EL ldence of u role IOI a cytochromr
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1131Goodhew.
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FEMS Microbiology Letters 135( 1996) 123- 129
A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase Anjana Ray, Huw D. Williams Department
ofBiology.
Imperial
College of Science, Technology
and Medicine,
*
Prince Consort Road.
LondonSW7 2BB. UK
Received 11September 1995; revised 30 October 1995; accepted 3 I October 1995
Abstract Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N, N, N’, N’-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway’s terminal oxidase is a quinol oxidase. Keywords:
Pseudomonas
aeruginosa;
Respiration; Cytochrome; Cytochrome oxidase; Haem
1. Introduction Pseudomonas aeruginosa is found in a very wide range of ecological niche ranging from soils and water sources to the lungs of patients with cystic fibrosis and the sites of bum wound infections [l]. These diverse environments will certainly differ in the availability and concentration of dissolved 0, and so respiratory adaptability may play a key role in the growth and survival of P. aeruginosa. One branch of the P. aeruginosa respiratory chain has a terminal oxidase that is highly resistant to the classical respiratory inhibitor potassium cyanide [2,3]. The molecular nature of this cyanide-insensitive oxidase (CIO) has not been elucidated but it is of interest because of the ability of P. aeruginosa to synthesise HCN, at
_ Corresponding author. Tel.: +44 (171) 594 5383; Fax: +44
( 17I) 584 2056; E-mail: [email protected]. 0378-1097/96/$12.00 SD/
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0378.1097(95)00440-8
1996
concentrations of 200-300 PM. Such levels would inhibit the cytochrome c oxidase, cytochrome co, which terminates a second branch of the aerobic respiratory chain [2-41. A third terminal oxidase, a cytochrome baa,, has been purified from a strain of P. aeruginosa and it is sensitive to cyanide (Iso = 50 PM), although there is no evidence yet for this oxidase in the well-characterised PA0 strains [2,5]. We are interested in the specific roles played by the terminal oxidases of this bacterium in its physiological adaptation to different environments. To this end we have isolated mutants defective in the CIO and cloned the complementary structural genes [2] and their sequence and characterisation will be published elsewhere. We are also investigating mutants defective in the cytochrome co-terminated pathway, both to aid genetic and physiological studies of this cytochrome c oxidase and to allow us to probe in greater detail the properties of the CIO-terminated
Federation of European Microbiological Societies. All rights reserved
pathway. The presence of c-type cytochromes in a respiratory chain allows the artificial electron donor N, N, N’, N’-tetramethyl-p-phenylenediamine (TMPD) to be oxidised to form an indophenol blue compound. Organisms that are unable to oxidise TMPD or do so poorly contain little or no cytochrome c in their aerobic respiratory chains [6]. Therefore, oxidation of TMPD provides both a means of specifically assaying the electron flux to OZ through a cytochrome c oxidase and a useful screen for isolating mutants defective in these pathways [3,7]. We have isolated a number of TMPD oxidase-defective mutants of P. aeruginosa following transposon mutagenesis and it is the properties of one such mutant that we describe in this paper.
2. Materials
and methods
2. I. Bacterial strains, media and growth conditions Pseudomonas aeruginosa PA06049 met-901 1 amiE200 strA [8], referred to as the wild-type throughout this paper, was used for Tn5-7.51 mutagenesis. PA07720 is the ccm mutant strain derived by Tn 5-751 mutagenesis of PA06049, and PA0772 1 is the ccm mutant complemented with the pADD386-based recombinant replicon PARS. Escherichia coli ED8654 metB supE supF hsdM hsdR was transformed with pME9, a derivative of the IncP-I based-plasmid pME305, which carries Tn5751 and acts as its delivery vehicle [S]. P. aeruginosa was grown aerobically in Luria-Bertani (LB) agar and LB broth [9], nutrient agar and nutrient yeast broth (Nutrient broth supplemented with 0.5% yeast extract). The incubation temperature was usually 30°C for PA06049 and for strains carrying pME9. Incubation at 43°C (non-permissive temperature for pME9) was used to cure strains of the plasmid. E. coli strains were grown at 37°C in LB. Anaerobic growth of P. aeruginosa was on LB + 0.2% KNO, and incubation was at 30°C in an anaerobic jar (Gas Pak, Becton Dickinson Co.). As required, antibiotics were added at the following concentrations ( pg ml ’ ), for E. coli: tetracycline (Tc, 251, kanamycin (Km, 251, trimethoprim (Tp, IOO), ampicillin (Ap, 100) and streptomycin (Sm, 200); and for P. aeruginosa: carbenicillin (Cb, 500), Tc (1251, Km (300) and Tp (500).
2.2. Preparation
of membranes
Membranes were prepared as described previously [2] from cells grown in LB broth until late exponential phase (OD,,,,, = I .O>. 2.3. Transposon mutagenesis and TMPD oxidase-negatil,e mutants
screening
,fbr
P. aeruginosa PA06049 was mutagenised using the Tn5-derivative, Tn5-751 [8]. The E. coli strain ED8654/pME9 carries Tn5-751 on the mobilisable plasmid replicon pME9. This strain served as a donor strain for conjugation with PA06049. E. coli ED8654/pME9 was grown at 30°C in nutrient yeast broth containing Km and Tc, whereas the recipient PA06049 was grown at 43°C to reduce activity of its restriction system [8]. For mating 5 ml volumes of late exponential phase cultures of donor and recipient cultures were mixed, pelleted by centrifugation and resuspended in 0.1 ml of medium and spread onto a nutrient agar plate to allow conjugation. After 4 h at 30°C the cells from the conjugation plate were collected by resuspension in 2 ml of saline (0.9% (w/v) NaCI) and plated onto NA + Tp, Km and Sm, and incubated at 30°C for 2-3 days. Simultaneous selection for resistance to Tp and Km selects for the presence of Tn 5-751 and eliminates the possibility of isolating spontaneous mutants. Sm counter selects against the E. coli donor strain. About 30000 KmR TpR SmK exconjugants were screened for loss of cytochrome c oxidase activity. This test uses the ability of the cytochrome c oxidase pathway to oxidise the artificial electron donor TMPD to an indophenol blue compound. The test was done by replica plating the exconjugants onto filter paper and then spraying them with a 9 mM solution of TMPD. Colonies that failed to turn blue after 30 s were taken as potential cytochrome c oxidase-negative mutants. Putative mutants were grown at 43°C to promote loss of the donor plasmid pME9 and its loss was verified by plating on NA + Tc and NA + Cb. 2.4. In l~ilv cloning
of complementing
genes
P. aeruginosa chromosomal DNA capable of complementing the TMPD oxidase-negative phenotype was cloned using the in vivo cloning system of Darzins et al. [ IO,1 I], using the mini-D31 12 based
replicon pADD386. The pADD386based gene library was used to transduce the oxidase mutant to CbR and transductants were subsequently screened for recovery of the ability to oxidise TMPD as described above. 2.5. C_vtochrome analysis Analysis of the cytochrome content of cells and membranes, using reduced-minus-oxidised and carbon monoxide (CO)-difference spectra was carried out as previously described [2,12]. Haem staining of membrane proteins to analyse for proteins with covalently bound haem c first involved pretreatment of membrane samples to remove potential interference from b-type cytochromes as described by Goodhew et al. [ 131. The pellet was then dissolved in electrophoresis sample buffer before SDS-PAGE and gels were stained for haem containing proteins using tetramethylbenzidine (TMBZ) as described by Thomas et al. [ 141. 2.6. Meusurement
of respirator? activities
Measurement of substrate-dependent 0, uptake rates. cyanide titration of activities and cytochrome c oxidase activity was carried out as previously described [2]. Myxathiazol-insensitive respiration was determined as 0, uptake in the presence of 100 PM myxathiazol, a concentration known to inhibit electron transfer through bacterial cytochrome bc, complexes [ 15,211. TMPD oxidase activity was assayed in membranes by following the increase in absorbance at 520 nm because of oxidation of TMPD [3]. All enzyme activity data represent the mean values from two or three experiments and there was typically no more than 10% variation between experiments. Protein assays were carried out using the method of Markwell et al. [16].
ability to oxidise TMPD to an indophenol blue compound. Twelve Tn5-751 insertion mutants were found which failed to turn blue or showed only slight blue coloration within 30 s of TMPD treatment. TMPD oxidase-deficient mutants could result from a number of possible respiratory defects. These include mutation of the structural genes of an obligatory component of the cytochrome (’ oxidase-terminated pathway, such as those for a cytochrome c’ or the terminal oxidase itself. Alternatively, the phenotype could result from a defect in the synthesis or assembly of a haem prosthetic group or some other essential co-factor or from mutation of a regulatory gene. The properties of one of these TMPD oxidase mutants, PA07720. are the subject of the remdmder of this paper. The mutant was complemented using the in vivo cloning system for P. aeruginosa based upon bacteriophage D3112 [ 10.1 I]. Phagemid Iysates of the mini-D replicon pADD386 carrying randomly packaged fragments of the P. aeruginosa chromosome were used to transduce PA07720 to CbR and transductants were screened for acquisition of a TMPD oxidase-positive phenotype. One complementing clone. PARS, was isolated and chosen fat further study. Both PA06049 and the mutant PA07720 had similar growth rates in LB although the mutant grew to significantly lower final optical densities than the wild-type (I .3S and 0.87 for PA06049 and PA07720, respectively). However. the mutant PA07720 was unable to grow anaerobically with nitrate as its terminal electron acceptor. The complemented mutant strain PA0772 I had identical growth characteristics to the wild-type. The lower final growth yield of the mutant is consistent with the loss of a thermodynamically efficient respiratory pathway. The inability to grow anaarohically on nitrate containing medium indicates a pleiotropic respiratory defect, affecting both the aerobic and anaerobic respiratory chains. 3.2. Biochemicul
charucterisation
of the mutarlt
3. Results and discussion 3.1. Isolation and complementation of mutants of P. aeruginosa dqfectir-e in TMPD oxidase actiri? Approximately 30000 KmR TpR exconjugants of P. aeruginosa PA06049 were screened for their
The only clearly demonstrated cytochrome c oxidase of P. aeruginosa PA06049 is cytochrome co [3,17]. The activity of the cytochrome co-terminated respiratory chain was measured by determining TMPD and cytochrome c oxidase activities. As can be seen in Table 1. membrane preparations from the
126
A. Ray, H.D. Williams/ FEMS Microbiology Letters 135 (19961 123-129
Table 1 TMPD and cytochrome PA0772 1
c oxidase
activities
Strain
in membranes
of P. aeruginosa wild-type,
TMPD oxidase activity (relative level)
PA06049 PA07720 PA07721
0.98 (I) ND ’ (0) 2.35 (2.4)
ccm ccm/pARS
a Given in pmol TMPD oxidised minb Given in pmol cytochrome c oxidised ’ ND, no activity detectable.
L
3,.
I,
I,,
550 Uavdeqth
1
MN) (nmn)
and complemented
mutant
Cytochrome c oxidase activity b (relative level)
’ (mg protein)-‘. min- ’ (mg protein)- ’
I,.......
SW
PA07720
54.7 (1) 7.8 (0.14) 60.6 (I.1 I)
mutant PA07720 had no TMPD oxidase activity and only 14% of the cytochrome c oxidase activity of the wild-type. pAR5 complemented these activities back to wild-type or higher levels. These results were expected from the plate assay phenotypes. Fig. 1 shows difference spectra of membranes of the various strains. In reduced-minus-oxidised spectra PA06049 shows an cz-maximum at 552 nm due to c-type cytochromes and a prominent shoulder at 558 nm due to cytochromes b (Fig. 1A). In contrast, in the mutant PA07720, the 552 nm peak corresponding to cytochromes c is absent and instead there is a maximum at 558 nm due to b- and possible o-type cytochromes (Fig. 1B). Approximate quantification of these spectral signals indicates that
500
A
mutant
1
554 Wavekgth
6W (nml
Fig. I. Difference spectra of membranes from wild-type, ccrn mutant and complemented mutant strains of P. aeruginosu. Reduced-minus-oxidised (A, B and C) and carbon monoxide (CO)difference spectra (D, E and F) were recorded of membranes from PA06049 (A, D>; PA07720 ccm (B, E) and PA0772 1 ccm /PARS (C, F). The vertical bar represents a change in absorbance (A A) of 0.06 (A, B and C), 0.012 (D and E) and 0.02 (F). All samples were at a protein concentration of 2 mg ml- ’
there is at least 70% decrease in the levels of c-type cytochromes in the mutant, Spectra of whole-cell samples gave similar results (data not shown). In the complemented mutant the cytochrome c signal at 552 nm reappears (Fig. 1C) and the levels of both cytochromes b and c are about 2.5 times higher than in the wild-type. The effect of the mutation on CO-binding cytochromes, and hence potential terminal oxidases, was determined from CO-difference spectra. PA06049 shows an asymmetric trough with minima at 550 and 558 nm probably largely due to the CO-binding haems c and o/b of the terminal oxidase, cytochrome co (Fig. 1D) [3,17]. The COdifference spectrum of the mutant is strikingly different (Fig. IE). Its spectrum is dominated by a large increase in the levels of a CO-binding cytochrome b with a deep trough at 558 nm. This would be explained if the mutation in PA07720 directly or indirectly led to the loss of a terminal respiratory pathway and there was a compensatory increase in the levels of a second pathway containing a CO-binding haem b/u. Complementation with pAR5 led to recovery of the wild-type spectral features, although the levels of CO-binding cytochrome c and cytochrome b were higher than in the wild-type (Fig. 1F). To establish more clearly whether the mutant had a pleiotropic defect in c-type cytochromes, crude extracts and membranes of the various strains were analysed by haem staining of gels following SDSPAGE. Membranes from the wild-type contained multiple haem staining bands (Fig. 2, lane 1). In contrast, no haem c-staining bands were present in the mutant PA07720 (Fig. 2, lane 2) while complementation by pAR5 led to recovery of all bands (Fig. 2, lane 3). So the spectral and haem staining gel data
127
A. Ray, H.D. Williams/ FEMS Microbiology Letters 135 (1996) 123-129
found that in TMPD- mutants of Rhodobucter cupsulutus, defective in genes subsequently shown to have roles in biogenesis of cytochrome c, had less than 20% of the spectrally detectable cytochrome c compared to the wild-type [19]. The fact that b-type cytochromes are still produced in P. aerwginosrr PA07720 indicates that the mutation does not have a pleiotropic defect in protoporphyrin IX synthesis. It suggests that the mutant is unable to form mature c-type cytochromes. The inability of the mutant to grow anaerobically is consistent with a cytochrome c maturation defect as a cytochrome c containing pathway is required for nitrite respiration, which occurs during anaerobic growth with nitrate [20]1. Haem groups of c-type cytochromes are covalantly attached to polypeptides via two covalent bonds formed between thiol groups of the polypeptide ‘and the vinyl groups of protohaem IX. Haem lyases, the enzymes which catalyse this reaction, have been identified in mitochondria [21] but none have yet been identified in bacteria, although it is assumed that they exist [ 181. Several other genes withipossible roles in cytochrome c biogenesis or maturation have been identified in bacteria (for review see [IS]). Sequencing of the complementing gene in pAR5 would obviously give some clues to its function if its sequence was homologous to known cytochrome c biogenesis genes. It has recently been suggested that a standard nomenclature be used in naming genes involved in these processes, namely that, they be called ccm genes for cytochrome c maturation [22]. While we have not as yet identified a specific gene
Fig. 2. Haem staining of polypeptides from membranes of wildtype, ccm mutant and complemented mutant strains. Membrane proteins were separated by SDS-PAGE and stained for haem with TMBZ as described in Materials and methods. Approximately 200 pg of protein was loaded in each lane. Lane 1, PA06049; lane 2, PA07720 ccm; lane 3, PA07721 ccm/pARS. The molecular mass markers at the side of lane I are, from top to bottom: 68, 43, 29, 18 and 14 kDa.
show that most if not all of the c-type cytochromes have been lost in the TMPD oxidase mutant. From these results it is clear that the mutation PA07720 has a pleiotropic effect on all haem c containing proteins in the membranes of P. aeruginosa. This P. aeruginosa mutant is reminiscent of mutants isolated, using similar screening procedures, in a number of different bacteria which have been shown to have defects in cytochrome c biogenesis. Kranz
Table 2 Comparison
of the respiratory
Strain
PA06049 PA07720 ccm PA0772 I ccm / pAR5
activities
of P. aeruginosa wild-type,
NADH-dependent Oz uptake a (relative levels)
CIO activity b (relative levels)
102 (1) 576 (5.6) 400 (3.9)
35 (1) 527 (15.1) 164 (4.7)
ccm mutant and complemented CIO activity
strains Myxathiazol-insensitive respiration ’ (%b)
(%o)
Myxathiazol-insensitive respiration ’ (relative levels)
34 91 41
49 (1) 421 (8.6) ND f
48 73 ND
’
min- ’ (mg protein)- ’ 0, uptake in the presence of 1 mM KCN, given in nmol 0, mm’ (mg protein)-‘. ’ CIO activity as a percentage of total NADH-dependent 0, uptake. d NADH-dependent 0, uptake in the presence of 100 PM myxathiazol, given in nmol 0, min- ’ (mg protein)’ Myxathiazol-insensitive respiration as a percentage of total NADH-dependent 0, uptake. ’ ND, not determined. a Given in nmol 0,
b NADH-dependent
’
or its function, we feel justified for the reasons given in referring to the mutated gene as a ccm gene. TMPD oxidase mutants of P. aeruginnsa have been isolated previously following chemical mutagenesis. Matsushita et al. [3] described the characterisation of such a mutant which, although it had no observable changes in cytochrome composition, was instrumental in demonstrating the presence of cyanide-insensitive respiration in this bacterium. While Yang [7] isolated a TMPD- mutant which did have lowered cytochrome c levels, the function of its cyanide-insensitive respiratory chain was not investigated. A mutant with a pleiotropic defect in c-type cytochromes allowed us to look at the role of c-type cytochromes in cyanide-insensitive respiration. The cyanide-insensitive oxidase (CIO) has not been isolated and it is not clear where its pathway branches off the main respiratory chain and hence whether its immediate electron donor is cytochrome c or a quinol. In the ccm mutant, malate-, succinate-, glucose- and gluconate-dependent 0, uptake rates were little changed (data not shown). However, NADHdependent O? uptake was more than five-fold higher in the mutant (Table 2). The activity was slightly lower in the complemented mutant but remained four-fold greater than the wild-type. Fig. 3 shows cyanide titration curves of NADH-dependent O? uptake. The wild-type had the expected biphasic titration curve [2,3] which indicates the presence in the membranes of at least two terminal oxidases with differing sensitivities to cyanide, with the more resistant respiration being due to the cyanide-insensitive oxidase [2]. In contrast, the ccm mutant PA07720 shows a monophasic titration curve with all respiration being cyanide-insensitive. This indicates that the entire NADH-dependent O2 uptake of this mutant is due to increased activity of the (JO-terminated pathway that corresponds to a ISfold increase in CIO activity (Table 2). This result clearly shows that the ccm mutant, which has lost all c-type cytochromes, still has a fully functional CIO-terminated respiratory pathway. The loss of the cytochrome c oxidase pathway leads to a massive increase in CIO activity, possibly in an attempt to compensate for the loss of electron flux to 0, through an energetically efficient cytochrome c oxidase. Complementation of the ccm mutation led to recovery of the biphasic titration curve (Fig. 3) and similar proportion of respiration in
‘ool~ 80
1
\T
Cyanide Concentration (M) Fig. 3. The effect of KCN on NADH-dependent the membranes of P. rrerqirww polarographically 100%
activity
PA07720
strains.
as described in Materials values
are
cc,,? (0 ), PA0772
given
in
oxygen uptake of
Activities were measured Table
I L’UII/PARS
and methods. The 2.
PA06049
cm).
( A ).
this strain was cyanide-insensitive as in the wild-type. However, the actual levels of respiration were still much higher than in the wild-type and this seems due to greater activity of both the CIO and cyanidesensitive pathways (Table 2) although this was not correlated with an increase in cytochrome L’oxidase activity (Table 1). We determined the effect of myxathiazol, which inhibits electron transfer through the ubiquinone-cytochrome c oxidoreductase complex (cytochrome hi.,), on O? uptake. The ccnz mutant PA07720 had a more than eight-fold increase in myxathiazol-insensitive respiration that accounted for at least 70% of all respiratory activity in this strain. Therefore, the CIO terminates a myxathiazol-insensitive pathway and confirms our expectations of a terminal oxidase that is able to function without c-type cytochromes, that is, it is most probably a quinol oxidase. While the molecular nature of the CIO has not been established, it is interesting to note that in the ccm mutant, which showed a ISfold increase in CIO activity, the only distinct spectral signal becoming more prominent is that of CO-binding cytochromes h.
[ 101Darwin\.
Acknowledgements
A. and Casaadaban. M.J. (I 989) In viva cloning oi
P,iu~frk~~~~or~o.~ omr,~i~~o.sc~genes with Mini-D?
This research was supported by a grant from the SERC (GR/F82214) to H.D.W. We thank D. Haas for providing the TnS-751 mutagenesis system and A. Darzins for providing the mini-D-based in vivo cloning system.
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A.M..
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in P.\c,cfr(c//~io~i~i.\. Methods I-nly-
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H.D. and Poole. R.K.
The cytoch;rotnc\ 01
~~~c,rohoc~rc,~ I’[‘.~tC,l,~i~l,r~,) NCIB 641X. EL ldence of u role IOI a cytochromr
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1131Goodhew.
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