4.2.1 Comparison of P. chlororaphis subsp. aurantiaca B-162 and P. chlororaphis subsp. aurantiaca B-162/255 genomes
To make a prediction about mutations that could cause phenazine production changes it is important to identify specific SNPs and functions of products of corresponding genes. The obtained sequences of B-162 and B-162/255 strains were aligned and 32 mutations were identified. All of them were qualified as single nucleotide polymorphisms (SNP). Six of these SNPs located in intergenic regions could hardly affect Pseudomonas metabolism. Among the rest 26 SNPs, 8 SNPs failed to modify amino acids and 3 induced conserved replacements (Table 1). Thus it was decided to focus on the remaining 15 genes with potentially important SNPs.
It is known that 16S ribosomal RNA (16S rRNA) is the component of the 30S small subunit of prokaryotic ribosome that binds to the Shine-Dalgarno sequence during translation [17]. The mutation that caused change proline to serine at position 108 was not important. This amino acid located at the initial part of α-helix and hadn`t an influence on binding or active
Table 1
List of proteins that are encoded by genes with detected mutations in comparative analysis of P. chlororaphis subsp. aurantiaca B-162 and P. chlororaphis subsp. aurantiaca B-162/255 strains
Name of protein
|
Position of mutation in genome, bp
|
Kind of mutation
|
16s rRNA
|
29758
|
G->A (Pro->Ser)
|
MerR family DNA-binding transcriptional regulator
|
823359
|
G->A (Thr->Ile)
|
Hypothetical protein
|
1005953
|
C->G (Ser->Ser)
|
Rod shape-determining protein MreC
|
1028732
|
T->C (Ser->Pro)
|
BioD-like N-terminal domain / Phosphate acetyltransferase (EC 2.3.1.8)
|
1066431
|
G->A (Val-> Val)
|
Translation elongation factor LepA
|
1193213
|
G->A (Val-> Ile)
|
Prophage tail fiber protein
|
1380270
|
G->A (Val-> Ile)
|
3-carboxy-cis,cis-muconate cycloisomerase (EC 5.5.1.2)
|
1476654
|
G->A (Gly-> Asp)
|
Cytosine/purine/uracil/thiamine/allantoin permease family protein
|
1697326
|
G->A (Arg-> Arg)
|
Phenylalanine hydroxylase transcriptional activator PhhR
|
1758287
|
G->A (Met-> Ile)
|
ADP-ribosylglycohydrolase
|
1985343
|
G->A (Pro-> Pro)
|
Sigma-54-dependent transcriptional regulator
|
2820154
|
G->A (Gly-> Asp)
|
Hypothetical protein
|
2864629
|
G->A (Arg -> Arg)
|
Domain of unknown function / Regulator of nucleoside diphosphate kinase
|
3387091
|
G->A (Asn -> Asn)
|
Type I secretion system ATPase, ABC-type protease exporter, ATP-binding component PrtD/AprD
|
3633870
|
C->G (Gly -> Arg)
|
Transcriptional regulator MvaT, P16 subunit, putative
|
3794621
|
G->A (Gly -> Asp)
|
Isoquinoline 1-oxidoreductase beta subunit (EC 1.3.99.16)
|
4327280
|
G->A (Val -> Met)
|
BarA-associated response regulator UvrY (= GacA = SirA)
|
4440666
|
G->T (Ser -> Arg)
|
Transcriptional regulator (Sigma-54 dependent transcriptional regulator)
|
4452736
|
G->T (Glu -> Lys)
|
Uncharacterized MFS-type transporter
|
4775194
|
G->A (Phe -> Phe)
|
Hypothetical protein
|
4778132
|
G->A (Val -> Met)
|
DNA-directed RNA polymerase alpha subunit (EC 2.7.7.6)
|
6398527
|
T->A (Asn -> Ile)
|
Predicted signal transduction protein (histidine kinase)
|
6714169
|
G->A (Thr -> Ile)
|
Alginate regulatory protein AlgP, positive transcriptional regulator of AlgD
|
6843467
|
C->T (Ala -> Ala)
|
Uncharacterized protein (Thioredoxin)
|
6876184
|
G->A (Leu -> Phe)
|
DNA-binding protein with helix-turn-helix motif
|
6971830
|
G->A (Pro -> Ser)
|
Note: Grey-colored genes carry mutations which may potentially affect protein functions.
|
sites of this protein. Locations of these sites were determined with the aid of homologous sequence (accession number in UniProt Q88RR3).
According to Brown, N.L. et al. the MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognition. The few MerR-like regulators studied experimentally have been shown to activate suboptimal σ70-dependent promoters. The majority of regulators of this family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. A subgroup of the family activates transcription in response to metal ions [18]. The mutation that caused threonine to isoleucine replacement at position 51 occurred in helix-turn-helix domain according to homologous sequence (accession number in UniProt A0A379I5Y4) and could influence activity of this protein.
It is known that rod shape-determining protein MreC is responsible for cell-morphology and several antibiotics sensitivity [19]. According to homologous sequence (accession number in UniProt W6QQZ2) the mutation that altered serine to proline at position 40 could not change active site of this protein.
3-carboxy-cis,cis-muconate cycloisomerase is the enzyme that catalyzes the conversion reaction of 2-carboxy-2,5-dihydro-5-oxofuran-2-acetate to cis,cis-butadiene-1,2,4-tricarboxylate [20]. The mutation that caused substitution glycine to aspartic acid at position 255 occurred in α-helix region and could not affect active sites of the protein according to homologous sequence (accession number in UniProt Q9I6Q8).
PhhR is transcriptional regulator that induces a cluster of structural genes phhABC encoding phenylalanine hydroxylase, carbinolamine dehydratase and aromatic aminotransferase respectively [21, 22]. Thus, PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation and phenylalanine homeostasis. The mutation that replaced methionine with isoleucine at position 260 occurred in σ54-factor interaction domain of this protein according to homologous sequence (accession number in UniProt G8PZM7). Motif analysis of these sequences showed the location of the mutation inside of motif sequence (Fig. 2). Since it is known that 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is regulated by phenylalanine and DAHP synthase is the key enzyme of shikimic acid pathway that leads to phenazines production [23] it seems that SNP in this gene could probably cause changes in phenazine antibiotics biosynthesis by mutant strain B-162/255.
It is known that transcription by RNA-polymerase utilizing σ54-factor is regulated by a distinct class of positive activators – σ54-dependent family regulators. The activities of these regulators are modulated in response to a wide variety of environmental signals. Factors that modulate the expression or the activity of the regulatory protein in response to chemical and metabolic changes are ultimately responsible for determining the level of expression of σ54-dependent genes and hence the diverse bacterial functions which they encode [24]. According to homologous sequence (accession number in UniProt Q880V5) the mutation that provokes glycine to aspartic acid replacement at position 268 is far away from interaction site of this protein and can’t interfere with its regulatory aspects.
Type I secretion systems consist of three membrane-localized proteins: ABC transporter, a membrane fusion protein and a TolC-like outer membrane component [25]. Using these systems a variety of small molecules such as antibiotics and toxins are removed from cells of gram-negative bacteria [26]. The mutation at position 357 took place in ABC transporter domain of B-162/255 strain according to homologous sequence (accession number in UniProt G8Q331). Motif analysis also showed the location of the mutation inside of motif sequence (Fig. 3).
Transcriptional regulator MvaT is a negative controller of chaperone-usher pathway gene cluster responsible for the biofilm formation in Pseudomonas bacteria [27]. According to the literature [28] biofilm formation may influence on phenazine synthesis in bacteria of this genus. Well known that this transcriptional regulator governs type III secretion system by repressing gene expression [29]. The mutation that replaced glycine with aspartic acid at position 98 did not exert any effect on regulatory sites of this protein according to homologous sequence (accession number in UniProt Q4KB87).Nevertheless motif analysis showed the location of the mutation in the center of motif sequence (Fig. 4).
Isoquinoline 1-oxidoreductase catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with subsequent reduction of a suitable electron acceptor utilizing H2O as the source of oxygen atom incorporated into the substrate [30]. The discovered mutation converting valine to methionine at position 581 probably cannot produce any impact on binding or active sites of this protein. Location of these sites was established with help of homologous sequence (accession number in UniProt A0A098T303).
We also determined mutation in GacA-response regulator gene. In Pseudomonas spp. secondary metabolism is controlled by the GacS/GacA global regulatory system. It determines the production of metabolites with antimicrobial activity including phenazines [31]. The mutation resulting in serine to arginine substitution at position 107 occurred between regulatory domains. Locations of these domains were evaluated via mediation of homologous sequence (accession number in UniProt P32967). Motif analysis of these sequences showed the location of the mutation inside of motif sequence (Fig. 5). It seems that this SNP could cause phenazine antibiotics biosynthesis by mutant strain B-162/255.
Another SNP was found in the gene encoding sigma factor σ54-dependent transcriptional regulator. The alteration of glutamic acid to arginine at position 420 was not localized in the interaction site of this protein and can’t influence its regulatory features.
It is widely known that DNA-directed RNA-polymerase is the enzyme that involved in transcription [17]. The mutation inducting asparagine to isoleucine change at position 273 occurred in α-C-terminal domain, but this amino acid was not involved in formation any active sites of protein. Location of this domain was specified using the homologous sequence of RNA-polymerase (accession number in UniProt Q4K557).
Histidine kinases are multifunctional, transmembrane proteins plays a key role in signal transduction across the cellular membrane [32]. Moreover, some of them are engaged in bacterial biofilm formation. Biofilms are bacterial microcolonies that may surround the roots of plants and protect them from pathogenic fungi thanks to produced phenazines [1]. The mutation causing threonine to isoleucine change at position 178 took place in regulatory domain according to homologous sequence (accession number of reference sequence in UniProt A0A0X1SW14). Motif analysis of these sequences showed the location of the mutation inside of motif sequence (Fig. 6). Therefore, this mutation potentially could promote phenazine production in B-162/255 mutant strain.
Thioredoxins are small redox-active proteins discovered in all organisms [33]. They play an essential role in important biological processes, including redox signaling. Mutation that caused leucine to phenylalanine change at position 301 by-passed the signal region was found in B-162/255 strain. Location of this region was determined with the help of homologous sequence (accession number in UniProt A0A089YX54).
The last protein was annotated as DNA-binding protein with helix-turn-helix motif. These proteins usually have a regulatory function [17]. The discovered mutation caused change of proline to serine at position 63 and took place in regulatory domain. Location of this domain was determined based on homologous sequence (accession number in UniProt Q4K3L3).
4.2.2 Comparison of P. chlororaphis subsp. aurantiaca B-162/255 and P. chlororaphis subsp. aurantiaca B-162/17 genomes
On the next step of the research sequences of B-162/255 and B-162/17 strains were compared. As it was mentioned above phenazines production in strain B-162/17 is higher than in wild-type bacteria but lower than in strain B-162/255. However, its distinctive feature is a production of phenazines on minimal media. To understand reasons of initiating phenazine production on minimal media sequences of B-162/255 and B-162/17 strains were aligned and 19 mutations were found. 16 of them were qualified as single nucleotide polymorphisms (SNP). Two of these SNPs located in intergenic regions hardly could affect phenazines metabolism. Among the rest 14 SNPs revealed in annotated gene sequences, 5 SNPs failed to modify amino acids and 3 induced conserved replacements (Table 2). Thus it was decided to focus on remaining 5 genes with potentially important SNPs. In addition to SNPs 3 deletions and 2 insertions were also detected. Two of these deletions directly influenced genes sequences so that they were included in the future analysis. The rest deletions and insertions happened in the intergenic regions.
As well as in the previous case to make a prediction about mutations that could cause phenazine production changes we analyzed all genes with potentially important SNPs.
Mutations that can be seen in genes that encode phenylalanine hydroxylase transcriptional activator PhhR and type I secretion system ATPase, ABC-type protease exporter, ATP-binding component PrtD/AprD are reverse mutations and sequence of these genes in B-162/17 strain was the same as in wild-type B-162 strain. Given the likely contribution of these genes in phenazine biosynthesis regulation and transport, it can be assumed that these reverse mutations could influence on phenazine production reduction in B-162/17 comparing to B-162/255 in special production media. The rest mutations appeared in B-162/17 de novo.
Table 2
List of proteins that are encoded by genes with detected mutations in comparative analysis of P. chlororaphis subsp. aurantiaca B-162/255 and P. chlororaphis subsp. aurantiaca B-162/17 strains
Name of protein
|
Position of mutation in genome, bp
|
Kind of mutation
|
Cytochrome c oxidase polypeptide I (EC 1.9.3.1)
|
68730–68741
|
Deletion of ccatgatgctga-region
|
Hypothetical protein (Outer membrane autotransporter barrel domain)
|
1649557
1649565
|
C->T (Asp->Asn)
T->A (Gln-> Leu)
|
Phenylalanine hydroxylase transcriptional activator PhhR
|
1758287
|
A->G (Ile -> Met)
|
RND efflux system, inner membrane transporter KPN_02144
|
2468524
|
T->G (Val-> Gly)
|
Immune-responsive protein 1
|
3276536
|
G->A (Leu-> Leu)
|
Type I secretion system ATPase, ABC-type protease exporter, ATP-binding component PrtD/AprD
|
3633870
|
G->C (Arg ->Gly)
|
Hypothetical protein
|
5018979
|
T->C (Leu -> Pro)
|
Efflux ABC transporter, permease/ATP-binding protein
|
5203838
|
G->A (Ala -> Val)
|
Hypothetical protein
|
5319599
|
G->A (Val -> Val)
|
Arginine N-succinyltransferase, alpha subunit (EC 2.3.1.109)
|
5392805–5392824
|
Deletion of ttcgccgacgcccagggctt-region
|
Glutaredoxin-related protein
|
5679384
|
G->A (Thr -> Thr)
|
PTS system, glucose-specific IIA component (EC 2.7.1.199) / Phosphotransferase system, phosphocarrier protein HPr / Phosphoenolpyruvate-protein phosphotransferase of PTS system (EC 2.7.3.9)
|
5753338
|
G->T (Leu -> Met)
|
Hypothetical protein
|
5832784
|
G->A (Asp-> Asp)
|
Alginate regulatory protein AlgP, positive transcriptional regulator of AlgD
|
6843467
|
C->T (Ala -> Ala)
|
Note: Grey-colored genes carry mutations which may potentially affect protein functions.
|
Cytochrome c oxidase catalyzes electron transfer and proton translocation reactions across membranes [34]. This enzyme consists of several subunits. The mutation in B-162/17 strain that caused deletion of 12 nucleotides led to the loss of 4 amino acids without a frameshift. One of the α-helix was reduced but it did not influence the formation of metal-binding site positioned 10 amino acids to the right of the deleted region according to homologous sequence (accession number in UniProt Q9I426).
Outer membrane autotransporter barrel-domain is a structural domain found in some bacterial outer membrane proteins [35]. The domain always located at the C-terminal end of the protein forms a β-barrel structure. The barrel is oriented in the membrane so that the N-terminal end of the protein, termed the passenger domain, presents on the cell surface. Two SNPs were detected in this sequence but none of them can influence on binding or active sites of the protein according to homologous sequence (accession number in UniProt A0A370LAR4).
According to comprehensive enzyme information system BRENDA [36] arginine N-succinyltransferase is an enzyme that catalyzes the chemical reaction of condensation of succinyl-CoA and L-arginine. The mutation that caused deletion of 20 nucleotides led to the frameshift and massive sequence changes on the C-terminus. There is no data about regulatory sites locations on this terminus. However, according to homologous sequence (accession number in UniProt P80357) formation of β-strand is disturbed by the frame shift. Potentially, it could alter the activity and (or) substrate specificity.
Cabeen, M.T. et al. discovered that phosphotransferase system is a major mechanism used by bacteria for carbohydrates recognition and uptake. Gene ptsP encodes enzyme I of the nitrogen-regulated phosphotransferase system vital for cyclic-di-GMP production and for biofilm formation [37]. The mutation site that initiated glycine to aspartic acid change at position 843 is far from any regulatory domains of this protein according to homologous sequence (accession number of reference sequence in UniProt Q4K6X2); therefore, its influence on protein function is minor.