Increased Activity of Cystathionine β-Lyase Suppresses 2-Aminoacrylate Stress inSalmonella enterica

ABSTRACTReactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. InSalmonella enterica, disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5′-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in aridAmutant strain. Degradation of cystathionine by MetC was not required for suppression ofridAphenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion.IMPORTANCERidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5′-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolismin vivo.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

Applied and Environmental Microbiology ◽

10.1128/aem.00380-11 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4455-4464 ◽

Cited By ~ 48

Author(s):

M. H. Wilbrink ◽

M. Petrusma ◽

L. Dijkhuizen ◽

R. van der Geize

Keyword(s):

Mutant Strain ◽

Coenzyme A ◽

Side Chain ◽

Side Chains ◽

Rhodococcus Rhodochrous ◽

Content Type ◽

Coa Ligase ◽

Coenzyme A Ligase ◽

Sequential Inactivation

ABSTRACTThe actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e.,fadD17,fadD19,fadE26,fadE27, andro04690DSM43269, by gene inactivation studies in mutant strain RG32 ofRhodococcus rhodochrousDSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of fivekshAgene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion offadD19in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation offadD17in mutant RG32ΔfadD19also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essentialin vivorole in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with anin vivorole in sterol side chain degradation. The high similarity (67%) between the FadD19DSM43269and FadD19H37Rvenzymes further suggests that FadD19H37Rvhas anin vivorole in sterol metabolism ofMycobacterium tuberculosisH37Rv.

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Mutations of the Listeria monocytogenes PeptidoglycanN-Deacetylase andO-Acetylase Result in Enhanced Lysozyme Sensitivity, Bacteriolysis, and Hyperinduction of Innate Immune Pathways

Infection and Immunity ◽

10.1128/iai.00077-11 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3596-3606 ◽

Cited By ~ 52

Author(s):

Chris S. Rae ◽

Aimee Geissler ◽

Paul C. Adamson ◽

Daniel A. Portnoy

Keyword(s):

Listeria Monocytogenes ◽

Transcriptional Responses ◽

Gram Positive ◽

Content Type ◽

Growth Defects ◽

Gram Positive Bacteria ◽

Host Cell Death

ABSTRACTListeria monocytogenesis a Gram-positive intracellular pathogen that is naturally resistant to lysozyme. Recently, it was shown that peptidoglycan modification by N-deacetylation or O-acetylation confers resistance to lysozyme in various Gram-positive bacteria, includingL. monocytogenes.L. monocytogenespeptidoglycan is deacetylated by the action ofN-acetylglucosamine deacetylase (Pgd) and acetylated byO-acetylmuramic acid transferase (Oat). We characterized Pgd−, Oat−, and double mutants to determine the specific role ofL. monocytogenespeptidoglycan acetylation in conferring lysozyme sensitivity during infection of macrophages and mice. Pgd−and Pgd−Oat−double mutants were attenuated approximately 2 and 3.5 logs, respectively,in vivo. In bone-marrow derived macrophages, the mutants demonstrated intracellular growth defects and increased induction of cytokine transcriptional responses that emanated from a phagosome and the cytosol. Lysozyme-sensitive mutants underwent bacteriolysis in the macrophage cytosol, resulting in AIM2-dependent pyroptosis. Each of thein vitrophenotypes was rescued upon infection of LysM−macrophages. The addition of extracellular lysozyme to LysM−macrophages restored cytokine induction, host cell death, andL. monocytogenesgrowth inhibition. This surprising observation suggests that extracellular lysozyme can access the macrophage cytosol and act on intracellular lysozyme-sensitive bacteria.

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Fosfomycin and Tobramycin in Combination Downregulate Nitrate Reductase GenesnarGandnarH, Resulting in Increased Activity against Pseudomonas aeruginosa under Anaerobic Conditions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00750-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5406-5414 ◽

Cited By ~ 15

Author(s):

Gerard McCaughey ◽

Deirdre F. Gilpin ◽

Thamarai Schneiders ◽

Lucas R. Hoffman ◽

Matt McKevitt ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Nitrate Reductase ◽

Growth Curves ◽

Anaerobic Growth ◽

Anaerobic Conditions ◽

Content Type ◽

Transposon Mutants ◽

Increased Activity ◽

Increased Susceptibility

ABSTRACTThe activity of aminoglycosides, which are used to treatPseudomonas aeruginosarespiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lungin vivo. In contrast, a 4:1 (wt/wt) combination of fosfomycin and tobramycin (F:T), which is under investigation for use in the treatment of CF lung infection, has increased activity againstP. aeruginosaunder anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests, including nitrate utilization assays, growth curves, and susceptibility testing. Notably, growth in subinhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (P< 0.05) nitrate reductase genesnarGandnarH, which are essential for normal anaerobic growth ofP. aeruginosa. Under anaerobic conditions, F:T significantly decreased (P< 0.001) nitrate utilization inP. aeruginosastrains PAO1, PA14, and PA14lasR::Gm, a mutant known to exhibit increased nitrate utilization. A similar effect was observed with two clinicalP. aeruginosaisolates. Growth curves indicate that nitrate reductase transposon mutants had reduced growth under anaerobic conditions, with these mutants also having increased susceptibility to F:T compared to the wild type under similar conditions. The results of this study suggest that downregulation of nitrate reductase genes resulting in reduced nitrate utilization is the mechanism underlying the increased activity of F:T under anaerobic conditions.

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Methionine Limitation Impairs Pathogen Expansion and Biofilm Formation Capacity

Applied and Environmental Microbiology ◽

10.1128/aem.00177-19 ◽

2019 ◽

Vol 85 (9) ◽

Cited By ~ 4

Author(s):

A. Jochim ◽

T. Shi ◽

D. Belikova ◽

S. Schwarz ◽

A. Peschel ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Human Serum ◽

Bacterial Infections ◽

Bacterial Species ◽

Biosynthesis Pathway ◽

Methionine Biosynthesis ◽

Content Type

ABSTRACTMultidrug-resistant bacterial pathogens are becoming increasingly prevalent, and novel strategies to treat bacterial infections caused by these organisms are desperately needed. Bacterial central metabolism is crucial for catabolic processes and provides precursors for anabolic pathways, such as the biosynthesis of essential biomolecules like amino acids or vitamins. However, most essential pathways are not regarded as good targets for antibiotic therapy since their products might be acquired from the environment. This issue raises doubts about the essentiality of such targets during infection. A putative target in bacterial anabolism is the methionine biosynthesis pathway. In contrast to humans, almost all bacteria carry methionine biosynthesis pathways which have often been suggested as putative targets for novel anti-infectives. While the growth of methionine auxotrophic strains can be stimulated by exogenous methionine, the extracellular concentrations required by most bacterial species are unknown. Furthermore, several phenotypic characteristics of methionine auxotrophs are only partly reversed by exogenous methionine. We investigated methionine auxotrophic mutants ofStaphylococcus aureus,Pseudomonas aeruginosa, andEscherichia coli(all differing in methionine biosynthesis enzymes) and found that each needed concentrations of exogenous methionine far exceeding that reported for human serum (∼30 µM). Accordingly, these methionine auxotrophs showed a reduced ability to proliferate in human serum. Additionally,S. aureusandP. aeruginosamethionine auxotrophs were significantly impaired in their ability to form and maintain biofilms. Altogether, our data show intrinsic defects of methionine auxotrophs. This result suggests that the pathway should be considered for further studies validating the therapeutic potential of inhibitors.IMPORTANCENew antibiotics that attack novel targets are needed to circumvent widespread resistance to conventional drugs. Bacterial anabolic pathways, such as the enzymes for biosynthesis of the essential amino acid methionine, have been proposed as potential targets. However, the eligibility of enzymes in these pathways as drug targets is unclear because metabolites might be acquired from the environment to overcome inhibition. We investigated the nutritional needs of methionine auxotrophs of the pathogensStaphylococcus aureus,Pseudomonas aeruginosa, andEscherichia coli. We found that each auxotrophic strain retained a growth disadvantage at methionine concentrations mimicking those availablein vivoand showed that biofilm biomass was strongly influenced by endogenous methionine biosynthesis. Our experiments suggest that inhibition of the methionine biosynthesis pathway has deleterious effects even in the presence of external methionine. Therefore, additional efforts to validate the effects of methionine biosynthesis inhibitorsin vivoare warranted.

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Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

Infection and Immunity ◽

10.1128/iai.01377-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1334-1340 ◽

Cited By ~ 12

Author(s):

Nelly Leung ◽

Antonella Gianfelice ◽

Scott D. Gray-Owen ◽

Keith Ireton

Keyword(s):

Listeria Monocytogenes ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Bacterial Pathogen ◽

Adaptor Protein ◽

Single Amino Acid ◽

Wild Type ◽

Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

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Certhrax Is an Antivirulence Factor for the Anthrax-Like OrganismBacillus cereusStrain G9241

Infection and Immunity ◽

10.1128/iai.00207-18 ◽

2018 ◽

Vol 86 (6) ◽

Cited By ~ 2

Author(s):

Yuliya I. Seldina ◽

Courtney D. Petro ◽

Stephanie L. Servetas ◽

James M. Vergis ◽

Christy L. Ventura ◽

...

Keyword(s):

Mutant Strain ◽

Protective Antigen ◽

Lethal Factor ◽

Toxin Gene ◽

Healthy Human ◽

Content Type ◽

Toxin Binding ◽

Edema Factor ◽

Capsule Locus

ABSTRACTBacillus cereusG9241 caused a life-threatening anthrax-like lung infection in a previously healthy human. This strain harbors two large virulence plasmids, pBCXO1 and pBC210, that are absent from typicalB. cereusisolates. The pBCXO1 plasmid is nearly identical to pXO1 fromBacillus anthracisand carries genes (pagA1,lef, andcya) for anthrax toxin components (protective antigen [called PA1 in G9241], lethal factor [LF], and edema factor [EF], respectively). The plasmid also has an intact hyaluronic acid capsule locus. The pBC210 plasmid has a tetrasaccharide capsule locus, a gene for a PA1 homolog called PA2 (pagA2), and a gene (cer) for Certhrax, an ADP-ribosyltransferase toxin that inactivates vinculin. LF, EF, and Certhrax require PA for entry into cells. In this study, we asked what role PA1, PA2, LF, and Certhrax play in the pathogenicity of G9241. To answer this, we generated isogenic deletion mutations in the targeted toxin gene components and then assessed the strains for virulence in highly G9241-susceptible (A/J) and moderately G9241-sensitive (C57BL/6) mice. We found that full virulence of G9241 required PA1 and LF, while PA2 contributed minimally to pathogenesis of G9241 but could not functionally replace PA1 as a toxin-binding subunitin vivo. Surprisingly, we discovered that Certhrax attenuated the virulence of G9241; i.e., a ΔcerΔlefmutant strain was more virulent than a Δlefmutant strain following subcutaneous inoculation of A/J mice. Moreover, the enzymatic activity of Certhrax contributed to this phenotype. We concluded that Certhrax acts as an antivirulence factor in the anthrax-like organismB. cereusG9241.

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RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans

Journal of Bacteriology ◽

10.1128/jb.00497-17 ◽

2017 ◽

Vol 199 (24) ◽

Cited By ~ 10

Author(s):

C. J. Kovacs ◽

R. C. Faustoferri ◽

R. G. Quivey

Keyword(s):

Cell Wall ◽

Stress Tolerance ◽

Streptococcus Mutans ◽

Mutant Strain ◽

Galleria Mellonella ◽

Infection Model ◽

Critical Function ◽

Content Type ◽

The Impact

ABSTRACT Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans. IMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

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Identification of Genes Important for Growth of Asymptomatic Bacteriuria Escherichia coli in Urine

Infection and Immunity ◽

10.1128/iai.00473-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3179-3188 ◽

Cited By ~ 22

Author(s):

Rebecca M. Vejborg ◽

Mari R. de Evgrafov ◽

Minh Duy Phan ◽

Makrina Totsika ◽

Mark A. Schembri ◽

...

Keyword(s):

Escherichia Coli ◽

Human Urine ◽

Asymptomatic Bacteriuria ◽

Genetic Determinants ◽

Content Type ◽

E Coli ◽

Growth Defects ◽

Cell Densities ◽

Tract Infections

ABSTRACTEscherichia coliis the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenicE. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU)E. colistrains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABUE. colican persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABUE. colistrain 83972 and the clinical ABUE. colistrain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB,argE,argC,purA,metE, andilvC), and site-specific mutants were subsequently constructed inE. coli83972 andE. coliVR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented intransas well as by supplementation with the appropriate amino acid or nucleobase. When assessedin vivoin a mouse model,E. coli83972carABand 83972argCshowed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metEand 83972ilvCdid not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABUE. coliin human urine.

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BPSS1504, a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.01544-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2006-2015 ◽

Cited By ~ 18

Author(s):

Verena Hopf ◽

André Göhler ◽

Kristin Eske-Pogodda ◽

Antje Bast ◽

Ivo Steinmetz ◽

...

Keyword(s):

Mutant Strain ◽

Burkholderia Pseudomallei ◽

Plaque Assay ◽

Giant Cells ◽

Host Cells ◽

Wild Type ◽

Secretion Systems ◽

Content Type ◽

Type Vi Secretion

ABSTRACTBurkholderia pseudomalleiis a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide.B. pseudomalleiharbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified aB. pseudomalleitransposon mutant defective in theBPSS1504gene that showed reduced plaque formation. TheBPSS1504locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis ofB. pseudomalleiin mammalian hosts. For further analysis, aB. pseudomalleiBPSS1504deletion (BpΔBPSS1504) mutant and complemented mutant strain were constructed.B. pseudomalleilacking theBPSS1504gene was highly attenuated in BALB/c mice, whereas thein vivovirulence of the complemented mutant strain was fully restored to the wild-type level. TheBpΔBPSS1504mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lackinghcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS componentsvgrG,tssA, andhcp1, as well as the T6SS regulatorsvirAG,bprC, andbsaN, was not dependent onBPSS1504expression. However, secretion of Hcp1 was not detectable in the absence ofBPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

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